In situ non-invasive spectral discrimination between bone cell phenotypes used in tissue engineering

Raman micro-spectroscopy was used to discriminate between different types of bone cells commonly used in tissue engineering of bone, with the aim of developing a method of phenotypic identification and classification. Three types of bone cells were analysed: human primary osteoblasts (HOB), retroviral transfected human alveolar bone cells with SV40 large T antigen (SV40 AB), and osteoblast-like human osteosarcoma derived MG63 cell line. Unsupervised principal component analysis (PCA) and linear discriminant analysis (LDA) of the Raman spectra succeeded in discriminating the osteosarcoma derived MG63 cells from the non-tumour cells (HOB and SV40 AB). No significant differences were observed between the Raman spectra of the HOB and SV40 AB cells, confirming the biochemical similarities between the two cell types. Difference spectra between tumour and non-tumour cells suggested that the spectral discrimination is based on the fact that MG63 osteosarcoma derived cells are characterised by lower concentrations of nucleic acids and higher relative concentrations of proteins compared to the non-tumour bone cells. A supervised classification model (LDA) was built and showed high cross-validation sensitivity (100%) and specificity (95%) for discriminating the MG63 cells and the non-tumour cells, with 96% of the cells being correctly classified either as tumour or non-tumour derived cells. This study proves the feasibility of using Raman spectroscopy to identify in situ phenotypic differences in living cells.     

Site-specific mutations in the D1 polypeptide affect the susceptibility of Synechocystis 6803 cells to photoinhibition

Photoinhibition of photosystem II in the cyanobacterium Synechocystis 6803 was followed after site-specific mutagenesis of the D1 polypeptide. Mutations were created in the stromal/cytosolic loop connecting helices D and E. Two mutations E243K and CA1, a deletion of the three glutamates 242–244 and a substitution Q241H, were made in the putative cleavage area of the D1 polypeptide. A third mutation E229D was made in the PEST-like sequence. Mutants and control cells were illuminated and F_V/F_M was recorded. Compared to the control, the mutants were less photoinhibited. Fluorescence relaxation after a single flash was delayed in CA1. Restoration of F_V/F_M after photoinhibition in the mutants was totally dependent on protein synthesis while control cells were able to recover partially also when protein synthesis was inhibited. In addition, the protein synthesis-dependent recovery of CA1 was slowed down. Our results indicate a correlation between the mutated amino acids and photoinhibition of photosystem II.     

The effects of constant and changing tempertures on the development of eggs of the freshwater snail Lymnaea auricularia (L.)

The rate of development of Lymnaea auricularia eggs was studied at various constant temperatures between 10° and 36°C. Development was accelerated as the temperature increased and at 36°C the eggs failed to develop. Spring eggs showed differences in their rate of development when compared with summer eggs when measured at similar tempertures.

Both spring and summer eggs were more than 90% fertile. Hatching success was high at temperatures between 10° and 30° (100%–82/9%); while at 34°C it was reduced to 60.6% for spring eggs. It was above 87% at temperatures between 10° and 34°C but it dropped to 62.3% at 36°C for summer eggs.

In one regularly changing temperature experiment a significant acceleration (P < .05) was found. In two others there was no significant difference beween predicted and observed egg durations. In one suddenly changing temperature regime (1 day at 20°, 1 day at 30° and so on) a huge retardation of development was found. In the other suddenly changing experiment (1 day at 15°, 1 day at 25°) no significant difference was found.

The exposure of eggs to extreme temperature (4°C, freezing and 4°C caused a retardation in the race of subsequent development of eggs at 25°C.     

X-ray structure of Na-ASP-2, a pathogenesis-related-1 protein from the nematode parasite, Necator americanus, and a vaccine antigen for human hookworm infection

Human hookworm infection is a major cause of anemia and malnutrition of adults and children in the developing world. As part of on-going efforts to control hookworm infection, The Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective L3 larval stages of the parasite, including a family of pathogenesis-related (PR) proteins known as the Ancylostoma-secreted proteins (ASPs). A novel crystal structure of Na-ASP-2, a PR-1 protein secreted by infective larvae of the human hookworm Necator americanus, has been solved to resolution limits of 1.68 A and to an R-factor of 17% using the recombinant protein expressed in and secreted by Pichia pastoris. The overall fold of Na-ASP-2 is a three-layer alphabetaalpha sandwich flanked by an N-terminal loop and a short, cysteine-rich C terminus. Our structure reveals a large central cavity that is flanked by His129 and Glu106, two residues that are well conserved in all parasitic nematode L3 ASPs. Na-ASP-2 has structural and charge similarities to chemokines, which suggests that Na-ASP-2 may be an extra-cellular ligand of an unknown receptor. Na-ASP-2 is a useful homology model for NIF, a natural antagonistic ligand of CR3 receptor. From these modeling studies, possible binding modes were predicted. In addition, this first structure of a PR-1 protein from parasitic helminths may shed light on the molecular basis of host-parasite interactions.     

Serum-free generation and quantification of functionally active Leukemia-derived DC is possible from malignant blasts in acute myeloid leukemia and myelodysplastic syndromes

Functional dendritic cells (DC) are professional antigen presenting cells (APC) and can be generated in vitro from leukemic cells from acute myeloid leukemia AML patients, giving rise to APC of leukemic origin presenting leukemic antigens (DC_leu). We have already shown that DC can be successfully generated from AML and myeloplastic syndromes (MDS) cells in serum-free standard medium (X-vivo + GM-CSF + IL-4 +TNF + FL) in 10–14 days. In this study, we present that DC counts generated from mononuclear cells (MNC) varied between 20% (from 55 MDS samples), 34% (from 100 AML samples) and 25% (from 38 healthy MNC samples) medium. Between 53% and 58% of DC are mature CD83⁺ DC. DC harvests were highest in monocytoid FAB types (AML-M4/M5, MDS-CMML) and independent from cytogenetic risk groups, demonstrating that DC-based strategies can be applied for patients with all cytogenetic risk groups. Proof of the clonal derivation of DC generated was obtained in five AML and four MDS cases with a combined FISH/immunophenotype analysis (FISH-IPA): The clonal numerical chromosome aberrations of the diseases were regularly codetectable with DC markers; however, not with all clonal cells being convertible to leukemia-derived DC_leu (on average, 53% of blasts in AML or MDS). To the contrary, not all DC generated carried the clonal aberration (on average, 51% of DC). In 41 AML and 13 MDS cases with a suitable antigen expression, we could confirm FISH-IPA data by Flow cytometry: although DC_leu are regularly detectable, on average only 57% of blasts in AML and 64% of blasts in MDS were converted to DC_leu. After coculture with DC in mixed lymphocyte reactions (MLR), autologous T cells from AML and MDS patients proliferate and upregulate costimulatory receptors. The specific lysis of leukemic cells by autologous T cells could be demonstrated in three cases with AML in a Fluorolysis assay. In six cases with only few DC_leu or few vital T cells available after the DC/MLR procedure, no lysis of allogeneic or autologous leukemic cells was seen, pointing to the crucial role of both partners in the lysis process. We conclude: (1) the generation of DC is regularly possible in AML and also in MDS under serum-free conditions. (2) Clonal/leukemia-derived DC_leu can be regularly generated from MDS and AML-MNC; however, not with all blasts being converted to DC_leu and not all DC generated carrying leukemic markers. We recommend to select DC_leu for vaccinations or ex vivo T-cell activations to avoid contaminations with non-converted blasts and non-leukemia-derived DC and to improve the harvest of specific, anti-leukemic T cells. DC and DC-primed T cells could provide a practical strategy for the immunotherapy of AML and MDS.     

Prognostic value of GLUT-1 expression in ovarian surface epithelial tumors: a morphometric study

OBJECTIVE: To investigate the reported increase in the expression of the glucose transporter GLUT-1 in borderline and malignant ovarian epithelial tumors and its relationship to prognosis. STUDY DESIGN: In this study, areas in which immunohistochemical membranous staining with GLUT-1 were most evident were selected, and the proportions of GLUT-1 expression in 46 benign, 11 borderline and 42 malignant cases of ovarian epithelial tumors were determined quantitatively with a computer and Zeiss Vision KS 400 3.0 (G?ttingen, Germany) for Windows (Microsoft, Redmond, Washington, U.S.A.) image analysis. RESULTS: GLUT-1 expression was determined in all borderline tumors (11 of 11) and in 97.6% of malignant tumors (41 of 42). No GLUT-1 expression was observed in benign tumors. The intensity of GLUT-1 staining was lower in borderline tumors than in malignant cases. This was statistically significant (p = 0.005). As differentiation in malignant tumors increased, proportions of GLUT-1 expression showed a relative increase, but this difference was not statistically significant (p = 0.68). CONCLUSION: When GLUT-1 expression in borderline and malignant ovarian epithelial tumors was analyzed against prognosis, no statistically significant difference was identified. Assessment of GLUT-1 expression using the image analysis program was more reliable, with higher reproducibility than in previous studies.     

Impact of <Emphasis Type="Italic">ERCC2</Emphasis> gene polymorphism on HIV-1 disease progression to AIDS among North Indian HIV patients

HIV/AIDS remains to be one of the killing diseases of mankind. Host genetic response is one of the factor which determine susceptibility to HIV and disease progression to AIDS. The aim of the present study was to evaluate the impact of ERCC2 Lyc ⁷⁵¹ Gln (excision repair cross complementing rodent repair deficiency, complementation group 2) polymorphism on HIV-1 susceptibility and disease progression to AIDS, as this gene has been reported to intervene in degrading retroviral cDNA before it integrates with the host DNA. This case control study included 300 HIV seropositive cases and an equal number of HIV seronegative controls. DNA was isolated from the blood samples of study subjects and genotyping of ERCC2 was conducted by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) method. The Gln/Gln genotype showed a significant variation between cases and controls (P = 0.047, OR 1.71, 95% CI 1.00–2.93), indicating a possible role of susceptibility in reference to controls and disease progression when compared within cases.     

Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae

Green fluorescent protein (GFP)-like pigments are responsible for the vivid colouration of many reef-building corals and have been proposed to act as photoprotectants. Their role remains controversial because the functional mechanism has not been elucidated. We provide direct evidence to support a photoprotective role of the non-fluorescent chromoproteins (CPs) that form a biochemically and photophysically distinct group of GFP-like proteins. Based on observations of Acropora nobilis from the Great Barrier Reef, we explored the photoprotective role of CPs by analysing five coral species under controlled conditions. In vitro and in hospite analyses of chlorophyll excitation demonstrate that screening by CPs leads to a reduction in chlorophyll excitation corresponding to the spectral properties of the specific CPs present in the coral tissues. Between 562 and 586 nm, the CPs maximal absorption range, there was an up to 50 % reduction of chlorophyll excitation. The screening was consistent for established and regenerating tissue and amongst symbiont clades A, C and D. Moreover, among two differently pigmented morphs of Acropora valida grown under identical light conditions and hosting subclade type C3 symbionts, high CP expression correlated with reduced photodamage under acute light stress.     

Determinants of Visceral Leishmaniasis: A Case-Control Study in Gedaref State,Sudan

Background

Improving knowledge on local determinants of visceral leishmaniasis (VL) is crucial to guide the development of relevant control strategies. This study aimed to identify individual and household level determinants of primary VL in 24 highly endemic villages of Tabarak Allah hospital’s catchment area, Gedaref State, Sudan.

Methods

From September 2012 to July 2013, in an unmatched case-control design, 198 patients with primary VL were compared to 801 controls free of VL symptoms and with a negative VL rapid test. Using random spatial sampling, controls were selected with a distribution of age, sex and village of residence proportionate to the distribution of the target population. Data were collected using a structured questionnaire.

Results

Children and men were at higher risk of VL. Reporting VL patient(s) in the household in the previous year was the strongest VL risk factor. In a multivariate analysis, VL risk increased with household size, sleep location (outside the yard, not in the farm), evening outdoor activities in the rainy season (playing, watching TV, radio listening), use of ground nut oil as animal repellent and of smoke of Acacia seyal as indoor repellent, presence of dogs in the yard at night, Acacia nilotica in the yard’s immediate surroundings and of a forest at eye range. VL risk appeared to decrease with the use of drinking water sources other than the village water tank, a buffer distance from the adjacent house yard, and with the presence of animals other than dogs in the yard at night. In contrast with previous studies, housing factors, mosquito-net use, black cotton soil, ethnicity, socioeconomic index, presence of Balanites aegyptica and Azadirachta indica in the yard were not independent VL determinants.

Discussion and conclusion

Although these results do not provide evidence of causality, they provide useful suggestions for guiding further intervention studies on VL preventive measures.     

Khat Chewing Habits in the Population of the Jazan Region,Saudi Arabia: Prevalence and Associated Factors

The use of khat (Catha edulis) is a major public health and social problem that is believed to be growing globally. The khat chewing habit is prevalent in all areas of the Jazan region, Kingdom of Saudi Arabia (KSA). However, few studies have been conducted at the community level to investigate the khat chewing habits in this area. This study was conducted with the aim of assessing the prevalence and predictors of khat chewing among the Jazan community population. A cross-sectional study was conducted with a sample (n = 4,500) of the Jizani population who attended primary heath care centers in Jazan region. The participants were selected using a two-stage cluster random sampling. A structured questionnaire was used for data collection. The overall lifetime prevalence of khat chewing was 33.2% (95% CI 31.8–34.7) and was significantly higher for males 42.2% (95% CI 40.4–43.9) than for females 11.3% (95% CI 9.6–13.1) (P < 0.001). Current khat chewers accounted for 28.7% (95% CI 27.4–30.1) of the population sampled; 36.9% (95% CI 35.2–38.6) of whom were males, which is a significantly higher percentage than the 8.7% (95% CI 7.3–10.4) of current khat chewers who were females (P < 0.001). The multivariate logistic regression analysis suggests that the most important independent predictors of khat chewing were having a friend who chewed khat (OR = 20.1, P < 0.001), participant''s smoking status (OR) = 3.9, P < 0.001), friend''s smoking status (OR = 2.2, P < 0.001), gender (OR = 2.2, P < 0.001) and educational level (OR = 1.5, P < 0.05). A large proportion of the Jizani populations chew khat. Government and non-governmental organizations NGOs should design and strengthen community prevention programs to curb the high prevalence of khat use.