Automated quantitative analysis of epithelial cell scatter

Epithelial cell scatter is a well-known in vitro model for the study of epithelial-mesenchymal transition (EMT). Scatter recapitulates many of the events that occur during EMT, including the dissociation of multicellular structures and increased cell motility. Because it has been implicated in tumor invasion and metastasis, much effort has been made to identify the molecular signals that regulate EMT. To better understand the quantitative contributions of these signals, we have developed metrics that quantitatively describe multiple aspects of cell scatter. One metric (cluster size) quantifies the disruption of intercellular adhesions while a second metric (nearest-neighbor distance) quantifies cell dispersion. We demonstrate that these metrics delineate the effects of individual cues and detect synergies between them. Specifically, we find epidermal growth factor (EGF), cholera toxin (CT) and insulin to synergistically reduce cluster sizes and increase nearest-neighbor distances. To facilitate the rapid measurement of our metrics from live-cell images, we have also developed automated techniques to identify cell nuclei and cell clusters in fluorescence images. Taken together, these studies provide broadly applicable quantitative image analysis techniques and insight into the control of epithelial cell scatter, both of which will contribute to the understanding of EMT and metastasis.Key words: cell scatter, EMT, automated image analysis     

Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide

Enterocytes exist in close association with tissue macrophages, whose activation during inflammatory processes leads to the release of nitric oxide (NO). Repair from mucosal injury requires the migration of enterocytes into the mucosal defect, a process that requires connexin43 (Cx43)-mediated gap junction communication between adjacent enterocytes. Enterocyte migration is inhibited during inflammatory conditions including necrotizing enterocolitis, in part, through impaired gap junction communication. We now hypothesize that activated macrophages inhibit gap junctions of adjacent enterocytes and seek to determine whether NO release from macrophages was involved. Using a coculture system of enterocytes and macrophages, we now demonstrate that "activation" of macrophages with lipopolysaccharide and interferon reduces the phosphorylation of Cx43 in adjacent enterocytes, an event known to inhibit gap junction communication. The effects of macrophages on enterocyte gap junctions could be reversed by treatment of macrophages with the inducible nitric oxide synthase (iNOS) inhibitor l-Lysine omega-acetamidine hydrochloride (l-NIL) and by incubation with macrophages from iNOS(-/-) mice, implicating NO in the process. Activated macrophages also caused a NO-dependent redistribution of connexin43 in adjacent enterocytes from the cell surface to an intracellular location, further suggesting NO release may inhibit gap junction function. Treatment of enterocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) markedly inhibited gap junction communication as determined using single cell microinjection of the gap junction tracer Lucifer yellow. Strikingly, activated macrophages inhibited enterocyte migration into a scraped wound, which was reversed by l-NIL pretreatment. These results implicate enterocyte gap junctions as a target of the NO-mediated effects of macrophages during intestinal inflammation, particularly where enterocyte migration is impaired.     

Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives

A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance the selectivity. Compound 11 resulted in IC(50) of 2.44, 2.5, and 2.7 microg/ml on MIAPaCa, PA-1, and SW620 cancer cell lines, respectively. Compound 38 resulted in IC(50) of 0.67 microg/ml on MIAPaCa cell line.     

NIH-supported national islet transplantation registry

An estimated 300,000 to 500,000 cases of type 1 diabetes exist today in the United States. Despite strict monitoring and attempts at control, people with type 1 diabetes still face the prospect of diminished health and earlier death than the general population. Islet transplantation offers an alternative to insulin usage and a potential treatment for type 1 diabetes mellitus. There are more than 30 islet transplant centers in the world focusing their efforts on the challenges and methods of this procedure. As the field of islet transplantation matures and the number of islet transplants performed increases, detailed analyses on factors that predict patient and graft survival are needed. This increased amount of data will allow for a better understanding of the safety and efficacy of islet transplantation. In response to the need for more complete information in the field, the National Institute of Diabetes and Digestive and Kidney Diseases is sponsoring the North American Collaborative Islet Transplant Registry (CITR). The mission of CITR is to expedite progress and promote safety in islet/β-cell transplantation through the collection, analysis, and communication of comprehensive and current data on all islet/β-cell transplants performed in North America. Compiling and analyzing data from all transplant centers in North America will accelerate the identification of both critical risk factors and key determinants of success, and thereby guide transplant centers in developing and refining islet/β-cell transplant protocols, leading to an advancement in the field of islet transplantation. Participation in CITR is voluntary, and more than 22 transplant centers have been invited to join. Seven centers are actively participating in CITR, with an additional 11 centers in the process of joining. Both an executive committee and a scientific advisory committee guide CITR. All islet transplants performed in North America since January 1, 1996, are captured by the CITR database. Through an electronic, Internet-based data capture system, quality control procedures, and minimization of duplicate efforts at the transplant center, the most relevant and succinct information are entered. From these data a comprehensive report will be published annually. In addition, special analyses will be performed and published periodically.     

Diagnostic considerations in prolymphocytes in pleural fluid: a case report

BACKGROUND: Prolymphocytes are nucleolated cells that are the defining features of the 2 chronic lymphoproliferative disorders, prolymphocytic leukemia (PLL) and chronic lymphocytic leukemia (CLL) with increased prolymphocytes. Prolymphocytes appear relatively unfamiliar in cytopathology practice, and, particularly when present in body fluids, may resemble blasts or adult T-cell leukemia/ lymphoma (ATLL) cells. CASE: A 32-year-old man, referred to us with a diagnosis of acute leukemia, presented with shortness of breath for 2 months and loss of appetite for 3 months. He had enlarged liver and spleen, 6 and 8 cm, respectively, below the costal margin and pleural effusion. The raised total leukocyte count chiefly comprised prolymphocytes that, especially in the pleural fluid, had prominent nucleoli and significant pleomorphism, raising the possibility of blasts or ATLL. CONCLUSION: Prolymphocytes in body fluids can be misinterpreted as blasts or even ATLL cells. Better awareness among cytopathologists about prolymphocytes and the disease states in which they occur, as well as insistence, in a clinical setting of leukemia, on interpreting the pleural fluid in relation to the clinical and laboratory findings, especially those of the peripheral blood and bone marrow, can prevent misdiagnosis. Equally importantly, immunophenotyping must be done in such situations.     

Regulation of Potassium-Dependent Kdp-ATPase Expression in the Nitrogen-Fixing Cyanobacterium Anabaena torulosa

The KdpB polypeptides in the cyanobacterium Anabaena torulosa were shown to be two membrane-bound proteins of about 78 kDa, expressed strictly under K(+) deficiency and repressed or degraded upon readdition of K(+). In both Anabaena and Escherichia coli strain MC4100, osmotic and ionic stresses caused no significant induction of steady-state KdpB levels during extreme potassium starvation.     

Location and interseasonal variation in flowering, propagule setting and propagule size in mangroves species of the family Rhizophoraceae

Three species of mangroves, Rhizophora stylosa, Rhizophora mangle (synonym R. samoensis) and Bruguiera gymnorhiza in the family Rhizophoraceae were studied to understand the flowering pattern, propagule development and the propagule size at maturity prior to dispersal from the mother plant. The study was conducted in the Wet and the Dry zones over two seasons in Viti Levu, the Main Island of Fiji. The flower number, number of propagules and propagule size at maturity were significantly different among three species and within species in the Dry and the Wet zones. Only 1–2% of total flowers in each species became mature propagules. This percentage was significantly lower in the Dry zone for all three species. Rhizophora stylosa produced the biggest size of propagules followed by Rhizophora mangle and Bruguiera gymnorhiza. Relatively longer and heavier propagules were recorded in the Wet zone and shorter and lighter in the Dry zone. Inter-seasonal differences were not significant for these characters. This could be mainly due to almost similar amount of rainfall, relative humidity and temperature regimes over two seasons within each zone.     

Reproductive exocrine and endocrine profiles and their seasonality in male langur monkeys (Presbytis entellus entellus)

The reproductive exocrine and endocrine profiles in male langurs are reported with an emphasis on seasonality. The animals showed positive response to electroejaculation throughout the year. The sperm concentration varied between 10–383 × 10⁶/ejaculation with wide fluctuations all through the year. No appreciable changes in the motility and percent live sperm were observed throughout the year. The levels of seminal fructose and magnesium remained unchanged throughout the year, while acid phosphatase showed wide fluctuations. Citric acid showed elevation during February and March and LDH showed elevated levels during April and May. The annual range of serum testosterone was 6–34 nMol/l with a peak during July. Cortisol ranged between 575–1587 nMol/l and prolactin ranged between 107–900 mU/l. Wide fluctuations were observed in hormonal levels. No seasonality was exhibited in the seminiferous tubule diameter, nuclear diameter of Sertoli cells and Leydig cells, and the cholesterol, glycogen, and sialic acid contents of testis. None of the parameters studied have shown any correlation with season. The results suggest that the male langurs lack seasonality in their reproductive exocrine and endocrine profiles and thus could be used as model for research in human reproduction.     

Dengue virus 2 capsid protein chaperones the strand displacement of 5′-3′ cyclization sequences

By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5′-3′ panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5′-3′ panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.