Evaluation of abdominal fat pad aspiration cytology and grading for detection in systemic amyloidosis

OBJECTIVE: To evaluate the diagnostic efficacy of abdominal fat pad aspiration cytology as a screening procedure for systemic amyloidosis and to assess the clinical usefulness of semiquantitative grading criteria of fat pad amyloid deposits. STUDY DESIGN: Aspiration cytology samples from 297 cases of abdominal fat pad were retrospectively analyzed for amyloid deposits. The smears were graded semiquantitatively. The deposits in the smears were compared with histologic evidence of amyloidosis in deeper tissues in 44 cases. RESULTS: Retrospective analysis of 297 cases of aspiration cytology revealed amyloid in 90 cases. Follow-up biopsies from deeper tissues in 44 cases showed presence of systemic amyloidosis in 13 cases. The sensitivity and specificity of abdominal fat pad fine needle aspiration cytology was 78% and 93%, respectively. The positive predictive value was 84% and negative predictive value 90%. CONCLUSION: Fat pad aspiration cytology is a useful screening procedure for diagnosis of systemic amyloidosis. Patients with grade 1 deposits should not undergo a toxic therapeutic regimen on the basis of fat pad cytology alone; histologic confirmation of visceral amyloid deposition in deeper tissue is advised. Patients with grades 2 and 3 deposits may undergo suitable therapy for amyloidosis.     

Implications of conformational flexibility,lipid binding,and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.     

Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion

Erythrocyte-binding antigen 140 (PfEBA-140) is a critical Plasmodium falciparum erythrocyte invasion ligand that engages glycophorin C on host erythrocytes during malaria infection. The minimal receptor-binding region of PfEBA-140 contains two conserved Duffy binding-like (DBL) domains, a fold unique to Plasmodium species. Here, we present the crystal structure of the receptor-binding region of PfEBA-140 at 2.4 Å resolution. The two-domain binding region is present as a monomer in the asymmetric unit, and the structure reveals novel features in PfEBA-140 that are likely determinants of receptor specificity. Analysis by small-angle x-ray scattering demonstrated that the minimal binding region is monomeric in solution, consistent with the crystal structure. Erythrocyte binding assays showed that the full-length binding region containing the tandem DBL domains is required for erythrocyte engagement, suggesting that both domains contain critical receptor contact sites. The electrostatic surface of PfEBA-140 elucidates a basic patch that constitutes a putative high-affinity binding interface spanning both DBL domains. Mutation of residues within this interface results in severely diminished erythrocyte binding. This study provides insight into the structural basis and mechanism of PfEBA-140 receptor engagement and forms a basis for future studies of this critical interaction. In addition, the solution and crystal structures allow the first identification of likely determinants of erythrocyte receptor specificity for P. falciparum invasion ligands. A complete understanding of the PfEBA-140 erythrocyte invasion pathway will aid in the design of invasion inhibitory therapeutics and vaccines.     

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.     

Influence of light on phycobiliprotein production in three marine cyanobacterial cultures

Complementary chromatic adaptation, a photomorphogenetic response, known to occur in many cyanobacteria, enables them to efficiently absorb prevalent wavelengths of light in the environment. In the present study, we have described the influence of light on phycobiliprotein production in three marine phycoerythrin producing cyanobacterial cultures, namely, Lyngbya sp. A09DM, Phormidium sp. A27DM and Halomicronema sp. A32DM. A comparative study (UV-visible overlay spectra and SDS-PAGE analyses) of phycobiliproteins purified from all the three cultures grown in white, yellow, red and green lights has been confirmed. White light was taken as control. Red and green lights were taken to check their effect on phycocyanin and phycoerythrin production, respectively. Yellow light was studied as its wavelength falls in between green and red light. Lyngbya sp. A09DM was found to be the best chromatically adapting cyanobacterium followed by Halomicronema sp. A32DM. These two cultures can be placed in group III chromatic adaptors. Phormidium sp. A27DM was the least chromatically adapting culture and can be placed in group II chromatic adaptors. The study signifies that even light plays an important role along with nutrient availability in adapting cultures to changing environmental conditions.