Identification of C-type lectin-domain proteins (CTLDPs) in silkworm Bombyx mori

C-type lectins (CTLs) represent a large family of proteins that can bind carbohydrate moieties normally in a calcium-dependent manner. CTLs play important roles in mediating cell adhesion and the recognition of pathogens in the immune system. In the present study, we have identified 23 CTL genes in domestic silkworm Bombyx mori. CTL-domain proteins (CTLDPs) are classified into three groups based on the number of carbohydrate-recognition domains (CRDs) and the domain architectures. These include twelve CTL-S (Single-CRD), six immulectins (Dual-CRD) and five CTL-X (CRD with other domains). We studied their phylogenetic features, analyzed the conserved residues, predicted tertiary structures, and examined the tissue expression profile and immune inducibility. Through bioinformatics analysis, we have putatively identified ten secretory and two cytoplasmic CTL-S; four secretory and two cytoplasmic immulectins; one secretory, one cytoplasmic and three transmembrane forms of CTL-X. Most B. mori CTLDPs form monophyletic groups with orthologs from Lepidoptera, Diptera, Coleoptera and Hymenoptera species. Immulectins of B. mori and Manduca sexta evolved from common ancestor genes perhaps due to gene duplication events of CTL-S ancestor genes. Homology modeling revealed that the overall structures of B. mori CTL domains are analogous to those of humans with a variable loop region. We examined the expression profile of CTLDP genes in naïve and immune-stimulated tissues. The expression and induction of CTLDP genes were related to the tissues and microorganisms. Together, our gene identification, sequence comparison, phylogenetic analysis, homology modeling and expression analysis laid a good foundation for the further studies of B. mori CTLDPs and comparative genomics.     

Molecular and functional characterization of pigeon (Columba livia) tumor necrosis factor receptor-associated factor 3

Tumor necrosis factor receptor-associated factor 3 (TRAF3) plays a key antiviral role by promoting type I interferon production. We cloned the pigeon TRAF3 gene (PiTRAF3) according to its predicted mRNA sequence to investigate its function. The 1704-bp full-length open reading frame encodes a 567-amino acid protein. One Ring finger, two TRAF-type Zinc fingers, one Coiled coil, and one MATH domain were inferred. RT-PCR showed that PiTRAF3 was expressed in all tissues, with relatively weak expression in the heart and liver. In HEK293T cells, over-expression of wild-type, △Ring, △Zinc finger, and △Coiled coil PiTRAF3, but not a △MATH form, significantly increased IFN-β promoter activity. Zinc finger and Coiled coil domains were essential for NF-κB activation. In chicken HD11 cells, PiTRAF3 increased IFN-β promoter activity and four domains were all contributing. R848 stimulation of pigeon peripheral blood mononuclear cells and splenocytes significantly increased expression of PiTRAF3 and the inflammatory cytokine genes CCL5, IL-8, and IL-10. These data demonstrate TRAF3's innate immune function and improve understanding of its involvement in poultry antiviral defense.     

Anti-dermatophyte activity of Leguminosae plants from Southern Brazil with emphasis on Mimosa pigra (Leguminosae)

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Mutations in rpoB gene and rifabutin susceptibility of multidrug-resistant Mycobacterium tuberculosis strains isolated in Australia.

Control of tuberculosis, the single largest killer among the infectious diseases, has been threatened by the emergence of multidrug-resistant Mycobacterium tuberculosis (MDRTB) infection due to the limited treatment options. Rifampicin (RIF) resistance is considered as a marker for MDRTB. The aim of this study was the detection of rpoB gene mutations and rifabutin resistance in MDRTB strains recently isolated in Australia by a line probe assay (INNO-LiPA Rif. TB, Innogenetics). Rifabutin and RIF susceptibility of 20 MDRTB and 16 RIF-sensitive M. tuberculosis complex clinical isolates were studied. The overall concordance of the line probe assay (LiPA) with phenotypic RIF susceptibility test was 96%. Seven distinct nucleotide substitutions were identified in 21 of 22 RIF-resistant isolates of diverse geographical origins, but in none of the RIF-sensitive strains. The majority (71%) of mutations occurred in the 526-533 codons and were associated with resistance to rifabutin and RIF. Of the RIF-resistant MDRTB strains, 18% appeared to be rifabutin-sensitive and produced delta S2 and delta S3 INNO-LiPA patterns. We conclude that amino acid substitutions at Asp516 and Ser522 in the rpoB gene in RIF-resistant M. tuberculosis predict rifabutin susceptibility for MDRTB. Use of the LiPA for RIF and rifabutin resistance may facilitate the rapid response required to limit the extent and severity of MDRTB transmission and infection.     

Mis-identification of factor inhibitors by diagnostic haemostasis laboratories: recognition of pitfalls and elucidation of strategies. A follow up to a large multicentre evaluation

AIMS: We previously reported the ability of diagnostic haemostasis facilities to identify coagulation factor abnormalities and inhibitors, through a large multi-centre study conducted on behalf of the Royal College of Pathologists of Australasia (RCPA) Quality Assurance Program (QAP). In the current report, additional data evaluation aims to (1) help identify the reasons behind the failures in inhibitor identification, (2) highlight the pitfalls in inhibitor testing, and (3) help elucidate some strategies for overcoming these problems and to assist in better identification and characterisation of inhibitors. METHODS: Forty-two laboratories blind tested a set of eight samples for the presence or absence of inhibitors. These included true factor inhibitors (FVIII and FV), and other samples that reflected potential pre-analytical variables (e.g., heparin contamination, serum, EDTA plasma, aged plasma) that might arise and complicate inhibitor detection or lead to false inhibitor identification. RESULTS: There was a wide scatter of inhibitor results, with false positive and false negative inhibitor identification, and mis-identification of inhibitors (e.g., FVIII inhibitor identified where FV inhibitor present). Further analysis of the pattern of reported laboratory results, including routine coagulation testing and factor profiles, allowed some additional interpretative power to provide strategies that will assist laboratories to improve the accuracy of inhibitor identification in the future. CONCLUSIONS: There are currently occasional lapses in factor inhibitor identification, which this report will hopefully help address.     

Identification and characterization of pufflectin from the grass pufferfish Takifugu niphobles and comparison of its expression with that of Takifugu rubripes

Pufflectin found in Takifugu rubripes (Tr pufflectin) is the first animal lectin reported to show sequence similarity to monocotyledonous plant lectins. In the present study, we identified and characterized an orthologous lectin from Takifugu niphobles (Tn pufflectin), a species closely related to T. rubripes. Tn pufflectin exhibits 86% identity to Tr pufflectin with two conserved mannose-binding domains. Tn pufflectin was mainly expressed in the skin, gills, brain, and muscles; however, it was expressed at a lower level in the other examined tissues. Recombinant Tn pufflectin, expressed by Escherichia coli, exhibited binding activity specific for d-mannose. The expression of pufflectin in the gills was much lower in T. niphobles than in T. rubripes; notably, the former and latter are resistant and susceptible, respectively, to the monogenean parasite Heterobothrium okamotoi, which parasitizes gills. This suggests that pufflectin might be utilized by the parasite for host recognition.     

Molecular methods in diagnosis and monitoring of haematological malignancies

The use of the polymerase chain reaction (PCR) was a revolutionary step in molecular biology, allowing for small amounts of genetic material to be amplified and studied. The advent of real-time PCR was a further refinement that led to reliable quantification of RNA and DNA. This allowed response monitoring and the detection of minimal residual disease, which proved to have important correlations with outcome in certain malignancies. The technology is indispensable for physicians and pathologists caring for oncology patients. In this article we will review the applications of molecular technology in the diagnosis and management of malignancies. Using chronic myeloid leukaemia (CML) as an example, technical aspects and clinical correlations will be discussed, with emphasis on the importance of quality assurance and standardisation to allow for comparability of results across laboratories. We will also examine emerging technologies that allow for high throughput and rapid turnaround of specimens and speculate how these would affect outcomes in future health care. The established and emerging molecular technologies have applications in many fields of oncology.     

Molecular biomarkers in malignant mesothelioma: state of the art

Malignant mesothelioma (MM) is an aggressive tumour affecting the mesothelial surfaces of the pleural and peritoneal cavities and, rarely, the pericardium and the tunica vaginalis testis. Despite a ban of asbestos in many industrialised nations, the present high incidence of MM is expected to continue, due to the long latency period between first asbestos exposure and occurrence of disease, making it an important health issue for the future. The diagnosis of MM can be difficult, both from a clinical and pathological perspective. It is not unusual for patients to undergo several medical investigations without definitive diagnosis early in their course of illness. Understandably, there is intense interest in the discovery of markers that can be assessed in pleural effusions, histological specimens, and serum to assist with the difficult early diagnosis of MM. Considering the primary aetiological role of asbestos, there is theoretically an easily identifiable target population for screening with a biomarker with adequate sensitivity and specificity or with a combination of biomarkers. In this review we focus on biomarkers that have been examined in the setting of either early diagnosis of MM in symptomatic patients or screening of asbestos-exposed individuals.     

Biosynthesis of collagen I,II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue

The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called ‘cell pellets’. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.     

Infectious diseases in paediatric pathology: experience from a developing country

Infectious and parasitic diseases have always challenged man. Although many of them are typically seen in some areas of the world and can be adequately managed by just improving socioeconomic status and sanitary conditions, they are still quite prevalent and may sometimes be seen outside their original geographical areas. Human migration due to different reasons, tourism, blood transfusion and solid organ transplantation has created new concerns for health professionals all over the world. If not for diagnostic purposes, at least these tropical and infectious diseases should be largely known because their epidemiology, pathogenesis, host/parasite interaction, inflammatory and reparative responses are quite interesting and teach us about human biology. Curiosity is inherent to pathology practice and so we are compelled to look for things other than tumours or degenerative diseases. This review focuses on infectious and parasitic diseases found in a developing country and brings up-to-date information on diseases caused by viruses (dengue, yellow fever), bacteria (typhoid fever, leprosy), parasites (Chagas' disease, cutaneous and visceral leishmaniasis, amoebiasis, Capillaria hepatica, schistosomiasis, cysticercosis) and caused by fungi (paracoccidioidomycosis, cryptococcosis, histoplasmosis) that may be useful for pathologists when facing somewhat strange cases from developing countries.     

Evolutionary history of the T cell receptor complex as revealed by small-spotted catshark (Scyliorhinus canicula)

In every jawed vertebrate species studied so far, the T cell receptor (TCR) complex is composed of two different TCR chains (α/β or γ/δ) and a number of CD3 subunits responsible for transmitting signals into the T cell. In this study, we characterised all of the TCR and CD3 genes of small-spotted catshark (Scyliorhinus canicula) and analysed their expression in a broad range of tissues. While the TCR complex is highly conserved across jawed vertebrates, we identified a number of differences in catshark, most notably the presence of two copies of both TCRβ and CD3γδ, and the absence of a functionally-important proline rich region from CD3ε. We also demonstrate that TCRβ has duplicated independently multiple times in jawed vertebrate evolution, bringing additional diversity to the TCR complex. This study reveals new insights about the evolutionary history of the TCR complex and raises new avenues for future exploration.     

Histological studies of neuroprotective effects of Curcuma longa Linn. on neuronal loss induced by dexamethasone treatment in the rat hippocampus

Long term exposure to dexamethasone (Dx) is associated with brain damage especially in the hippocampus via the oxidative stress pathway. Previously, an ethanolic extract from Curcuma longa Linn. (CL) containing the curcumin constituent has been reported to produce antioxidant effects. However, its neuroprotective property on brain histology has remained unexplored. This study has examined the effects of a CL extract on the densities of cresyl violet positive neurons and glial fibrillary acidic protein immunoreactive (GFAP-ir) astrocytes in the hippocampus of Dx treated male rats. It showed that 21days of Dx treatment (0.5 mg/kg, i.p. once daily) significantly reduced the densities of cresyl violet positive neurons in the sub-areas CA1, CA3 and the dentate gyrus, but not in the CA2 area. However, CL pretreatment (100 mg/kg, p.o.) was found to significantly restore neuronal densities in the CA1 and dentate gyrus. In addition, Dx treatment also significantly decreased the densities of the GFAP-ir astrocytes in the sub-areas CA1, CA3 and the dentate gyrus. However, CL pretreatment (100 mg/kg, p.o.) failed to protect the loss of astrocytes in these sub-areas. These findings confirm the neuroprotective effects of the CL extract and indicate that the cause of astrocyte loss might be partially reduced by a non-oxidative mechanism. Moreover, the detection of neuronal and glial densities was suitable method to study brain damage and the effects of treatment.     

Regulation of Nlrp3 inflammasome by dietary metabolites

The bidirectional communication between innate immune cells and energy metabolism is now widely appreciated to regulate homeostasis as well as chronic diseases that emerge from dysregulated inflammation. Macronutrients-derived from diet or endogenous pathways that generate and divert metabolites into energetic or biosynthetic pathways – regulate the initiation, duration and cessation of the inflammatory response. The NLRP3 inflammasome is an important innate sensor of structurally diverse metabolic damage-associated molecular patterns (DAMPs) that has been implicated in a wide range of inflammatory disorders associated with caloric excess, adiposity and aging. Understanding the regulators of immune-metabolic interactions and their contribution towards chronic disease mechanisms, therefore, has the potential to reduce disease pathology, improve quality of life in elderly and promote the extension of healthspan. Just as specialized subsets of immune cells dampen inflammation through the production of negative regulatory cytokines; specific immunoregulatory metabolites can deactivate inflammasome-mediated immune activation. Here, we highlight the role of energy substrates, alternative fuels and metabolic DAMPs in the regulation of the NLRP3 inflammasome and discuss potential dietary interventions that may impact sterile inflammatory disease.