A comprehensive understanding of the cytoskeleton can only be achieved by the combination of biochemical, cellular, and whole organism studies As a student I always marvelled at the sight of single cells in culture moving over artificial surfaces and exhibiting membrane ruffles and protrusions. However, while I found cultured cells fascinating I always wondered how cells are able to move and regulate their shape in the context of a whole organism where so many space constraints exist and where all cellular processes have to be tightly regulated. Some answers to my questions began to emerge in a paper written by Drosophila, which offers virtually unlimited possibilities as a model system for the genetic and molecular analysis of biological processes.The cytoskeleton is a meshwork of protein polymers extending throughout the cytoplasm. It not only provides structural support for the cell but also plays a central role in a range of dynamic processes from signalling to endocytosis and intracellular trafficking. A particularly clear example of this is the use of actin cytoskeleton as a “wool” for knitting multiple dynamic structures such as lamellae, filopodia, and stress fibres. These structures determine cell shape and also produce the driving force accompanying many types of cellular movements including muscle contraction and cell division. We know many details about some of the proteins that modulate the dynamics of actin in these structures. However, most of them have been found biochemically and their function has been elucidated primarily using in vitro and cell culture assays of actin assembly. What about these proteins in the context of a developing organism? How do cells generate a spatially and temporally ordered network of actin filaments represented at the tissue level? To answer these questions, we need to move to experimentally accessible multicellular organisms, such as Drosophila oogenesis. These cells have a simple polarised arrangement of actin filaments, which provides a useful system to study the spatial organisation of the actin cytoskeleton.Drosophila homologue of adenylyl cyclase-associated proteins), Enabled (Ena) and Abelson (Abl). These proteins had been well characterized in cell culture and in vitro studies, but little was known about their function in a developing organism. Clones of cells lacking CAP (Taking advantage of the ability to generate clones of cells lacking specific proteins, the authors identified new functional roles for actin regulators such as CAP (a king CAP , a proteIn contrast to the spatially restricted functions of CAP, Ena, and Abl, profilin and cofilin were shown to regulate actin filament formation throughout the cell cortex, a more global function that matches the results obtained in cell culture experiments. In summary, this study showed how proteins can organise actin in space and began to highlight some of the differences and similarities between cells in culture and in vivo. The functions revealed in the follicular epithelium were consistent with the roles previously shown in mammalian systems, but the experiments on intact tissue began to reveal a spatial and temporal functional dimension that could not have been observed in cell culture.These experiments could be expanded to large-scale screens , but thiDrosophila to screen a number of genes involved in signalling and cytoskeletal dynamics. They targeted 994 genes, of which 160 produced phenotypes in the experiment. The range of phenotypes varied from specific defects in the actin and tubulin cytoskeleton to others affecting cell cycle progression, cytokinesis, and cell shape. They also showed that only about 40% of the genes had similar loss-of-function phenotypes in both cell lines. This alone indicates an important limitation of many tissue culture experiments, since the same protein can have different effects depending on the cell type. Another valuable element of this work is that clustering of genes with similar phenotypes leads to the identification of pathways and networks of genes that are involved in cytoskeletal function.Drosophila cell line, studied the effects of proteins involved in the formation of lamellae. The authors looked at the effects of loss of function in 90 genes known to be involved in actin dynamics and the formation and activity of the lamella. As well as confirming the function of many proteins already known to play a role in this process, this analysis allowed them to find interactions between genes and to build genetic pathways.Drosophila embryos, as an example of how signalling pathways coordinate and regulate the activity of the cytoskeleton in the generation of shape and morphogenetic movements (Together these two studies reveal that RNAi screens in tissue culture can be a powerful tool for finding new functions of known and uncharacterized genes, and new relationships between genes. However, this is only the beginning, and the genes identified in this manner will have to be tested in vivo, in systems like that of Baum and Perrimon, where specific functions can be assessed in time and space within the confines of real organisms. The focus must be to understand how all these molecular events and regulation cascades operate in individual cells to contribute to the generation of changes in a whole individual. Increasingly, the attention of developmental biologists is being drawn from genes and their products towards cells . The futovements .


Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target.M. hominis PG21 genomic DNA. A fluorescence signal was measured for all 20 other M. hominis isolates, and melting curves analysis showed variations in the melting temperature in agreement with sequence variation in the region of the probes. There was no amplification of other mycoplasmal DNA and human DNA. Eighty-three patient cervical swab samples from infertile women were cultured for M. hominis in the BEa medium. Two of the samples (2.4%) were positive after 48 hours of incubation. The real-time PCR detected the same two samples positive, and the DNA concentrations in the clinical specimens were calculated to 37.000 copies/ml and 88.500 copies/ml, respectively.Real-time PCR was optimized to detect 10 copies of M. hominis in a closed system reducing the risk of contamination by amplicon carry-over.The results demonstrate that real-time PCR may prove to be a rapid alternative to the traditional cultivation method. Information on bacterial load in genital swabs can be obtained. The assay allowed detection of  Mollicutes and are distinguished phenotypically from other bacteria by their minute size and lack of a cell wall. Genetically they differ by having a small genome size and low G+C content {1} [Mycoplasmas are the smallest living prokaryotes known, capable of self-replication. They belong to the class Mycoplasma isolated was Mycoplasma hominis [M. hominis has also been isolated from the endometrium and fallopian tubes of 10% women with salpingitis. However, its role as a primary pathogen is doubtful since it co-exists with many other bacteria in BV [in vitro fertilization showed the presence of M. hominis only in 2.1% of the women [The first human  hominis . It is a hominis  found in hominis ,5. M. hoia in BV . Studieshe women .M. hominis has been found to cause wound, joint and central nervous system infections [M. hominis and indicated a need for rapid recognition. So far culture is most commonly used for detection of genital Mycoplasma, but it requires special handling, complex media, and cultivation positive samples need further testing to determinate the species cultivated [Isolation from other sites than the genitourinary tract has been reported. fections , it has fections  and casefections -15. Thosltivated . A case ltivated  showed tM. hominis from clinical samples. In addition it was very specific [Comparison between culture method and PCR has been performed and showed that a PCR assay was as sensitive as culture for detection of specific . An advaM. hominis by real-time PCR in HIV-positive patient swab samples, suggested the use of SYBR Green with primers targeting another housekeeping gene, the 16S rRNA gene [M. hominis minor sequence variation was observed [M. hominis isolates were available (see table gap) as target for development of a quantitative real-time PCR. Comparison of the DNA sequences from different M. hominis isolates showed, however, some small variations in the amplified DNA sequence [M. hominis LightCycler real-time PCR and tested it on clinical swab samples using specific hybridization probes for detection.A recent study designed for the detection of RNA gene . The 16Sobserved . Becausesequence . We deteThe principles of the system we used are based on two specific hybridization probes located internally to the amplification primers, each of them labelled with a different fluorescent dye . The DNAM. hominis PG21 type strain as a template for primers and probes. The primers and probes were designed with respect to conservation of the DNA sequence within the M. hominis gap gene [Mycoplasma genitalium gap gene (Accession no. U39710) . By use of the MOTIFS program the active site was predicted to consist of the amino acids: ASCTTNCL, located at nucleotides 451 to 474  were compared by calculation of the coefficients of variation (CV). The values of CV were between 3.8% and 6.7%  were spiked with 2 μl of PG21 DNA of 100 copies/μl and added to the reaction mix. As illustrated  is defined as the temperature at which half of a duplex-DNA becomes single-stranded [M. hominis isolates. Tm of PG21 DNA was 66°C and equal for high and low concentrations of DNA.Melting curve analysis can be used to determine the presence of non-specific amplification products. The melting temperature . Two μl of the samples were analyzed by the LightCycler PCR. As shown  were positive when examined by LightCycler PCR (2.4%). The copy numbers were measured to be 220 (for 83) and 530 (for 56) copies/μl respectively. The amount of DNA copies per ml in the original sample was calculated to be 37.000 for sample 83 and 88.500 for sample 56. The two quantification methods for estimating bacterial load in positive samples thus showed that the number of live Mycoplasma cells was 69% (patient 83) and 29% (patient 56) of what was found by PCR. The positive clinical samples showed melting temperature of 64°C corresponding to the second group, in which the majority of the M. hominis isolates were found. Reproducibility of the quantification of DNA in the clinical specimens was analyzed in 10 negative patient samples. Four μl of samples were spiked with 2 μl of PG21 DNA of known concentration (100 copies/μl). The six μl were added to the LightCycler PCR reaction. No inhibition was observed  also had the flat melting curve with the melting peak below 61°C fig.  and wereM. hominis DNA, we analysed proteinase K treated samples from those patients by the LightCycler PCR. None of the samples gave a positive fluorescence signal. After confirming that all samples were negative, we spiked 2 μl of each sample with 2 μl of PG21 DNA of 100 copies/μl, as the control for inhibition. None of the proteinase K treated samples showed inhibition. This clearly indicates that those samples were negative.To additionally confirm that samples which gave a slightly positive fluorescence signal in LightCycler PCR after DNeasy treatment were not containing M. hominis from cervical swab samples was developed. To our knowledge it is the first LightCycler PCR protocol where quantification of M. hominis is combined with melting curve assay. The LightCycler PCR reproducibility was able to detect down to ten copies/reaction of the genomic PG21 DNA. All other 20 M. hominis isolates were positive in the assay. Additionally, isolates 132 and 4712, which based on melting temperature belong to the two other groups than PG21, were used to document detection limits of M. hominis isolates. Similarly to PG21, the detection limits were 5 copies/μl, equals to 10 copies per reaction for both isolates.A rapid quantitative real-time PCR for detection of The average efficiency was high, but small differences were seen. This can be caused by different factors such as presence of inhibitors in the sample and treatment of the sample.Mycoplasma species with PBS. Additionally, a spiking assay where DNA from other mycoplasmas was used together with DNA from PG21 showed that there was no inhibition in the proteinase K treated samples.The artificially constructed samples consisting of PG21 DNA and coloured BEa and SP-4 media showed that these media inhibited the PCR reaction indicating that it was crucial to wash the pellets of other Since swab samples may contain PCR inhibitors we introduced the DNeasy procedure to additionally purify DNA from proteinase K treated clinical samples. Analysis of the ten negative clinical samples spiked with DNA from PG21 (100 copies/μl) showed no inhibition after DNeasy treatment.M. hominis isolates  were studied. All patients were undergoing hysteroscopy and transvaginal hydrolaparoscopy (culdoscopy)  or laparEndocervical specimens were obtained using a sterile chlamydial swab and the contents transferred immediately into a tube containing 2 ml of transport medium, SP-4 , containM. hominis, M. buccale, M. salivarium, M. orale, M. arthritidis, M. arginini, M. lipophilum, M. primatum were cultivated in 1.7 ml of broth BEa medium [U. urealyticum and U. parvum were grown in 1.7 ml of SU medium [M. pneumoniae and M. genitalium were grown in 100 ml of SP-4 medium [M. fermentans, M. pulmonis, M. hyorhinis were cultivated in 1.7 ml of BEg medium [M. bovis was cultured in BE medium [M. hominis, M. genitalium and M. pneumoniae, which were cultivated up to bigger volumes (100 ml), 500 μl of the colour changed cultures were then placed in 7 ml of the new medium, and harvested after the medium changed for the second time. One ml of logarithmic-phase culture was centrifuged in Eppendorf tubes at 20.000 × g for 30 min. Each pellet was washed twice by phosphate-buffered saline (PBS) and the pellets were stored at -70° prior to use.All isolates of a medium . U. ureaU medium . M. pneu4 medium  as descr4 medium . M. fermg medium . FinallyE medium . All culHuman Hep2 cells were cultured as described elsewhere .M. arginini, M. bovis, M. hyorhinis, M. pulmonis, M. primatum, M. lipophilum, M. buccale, M. salivarium, M. orale, M. arthritidis, U. urealyticum, U. parvum, M. pneumoniae and M. fermentans was extracted by suspending pellets in 160 μl TE buffer, and adding 40 μl 10 mg/ml proteinase K. The proteins were digested by incubation at 55°C for one hour, followed by inactivation of the enzyme by boiling for 10 minutes at 100°C.DNA from M. genitalium DNA was extracted and purified by Blood & Cell Culture DNA Mini Kit .The DNA from human Hep2 cells as well as M. hominis isolates was extracted as described [Genomic DNA from all escribed  and follescribed .Concentrations of DNA from isolates PG21, 132 and 4712, and human Hep2 DNA were calculated after measuring OD  at 260 and 280 nm with Unicam 8625 UV/VIS spectrometer .Fourfold dilution series of human Hep2 DNA purified by Blood & Cell Culture DNA Mini Kit  of initial concentration 8.000 copies/μl were prepared. Twenty-five μl of each dilution and DNA free double distilled water were then treated with DNeasy™ Tissue Kit  procedure. Samples were diluted twice, because of elution step with 50 μl of elute buffer, and therefore 4 μl instead of two of each sample were used in the LightCycler PCR run.M. hominis. To avoid overgrowth by other bacteria, present in the urogenital tract, a special mixture of antibiotics  was used. Twenty μl of the swab sample was placed in 1 ml of medium with Niels Friis antibiotics and incubated at 37°C. The BEa medium changed colour from orange to strawberry pink in 48 hours due to reduction of phenol red by arginine hydrolysis. Finally, samples were described as positive when it was possible to pass them further 2 times. Mycoplasma-genus-specific PCR was performed on the two positive cultures and the PCR products were sequenced. Sequencing reactions were carried out bidirectionally using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit  on the purified PCR products according to the instructions supplied by the manufacturer. Sequencing was performed on an ABI PRISM 377 DNA Sequencer .Broth BEa medium was used for culture of g, and to remove transportation medium the pellets were washed twice with PBS. The pellets were suspended in 49 μl TE buffer, and 1 μl 10 mg/ml Proteinase K was added, proteins were digested while incubating at 55°C for one hour, which was followed by inactivation of the enzyme by boiling for 10 minutes at 100°C. Finally, in order to remove possible inhibitors present in swab samples, DNA extraction with DNeasy™ Tissue Kit  was performed on the resulting solution without repeating the proteinase K treatment. Twenty-five μl of the proteinase K treated sample was diluted twice because of elution with 50 μl of elute buffer, 4 μl of each sample was used for the PCR. The filter pipette tips were used for all DNA preparation steps to reduce possibility of sample contamination.All 83 patient samples were treated identically. Briefly, three hundred μl of the original swab sample was subjected to microcentrifugation at 20.000 × M. hominis in patient swab samples was performed by titration in BEa growth medium. Two-fold dilutions were made in ELISA trays by adding 0.01 ml of the clinical sample to 0.19 ml of BEa medium. The plates were incubated at 37°C and reading was performed on the third day. Plates were left in incubator and observed for 3 additional days but no further change appeared. The last well with visible colour change was considered to contain one colour changing unit (CCU) allowing us to calculate the number of viable microorganisms in the original clinical sample.Estimation of the number of gap gene of Mycoplasma hominis type strain PG21 (Accession No. AJ243692). This gene belongs to the housekeeping genes and is therefore very conservative in all organisms. Primer and probe sequences and their locations are present in table gap gene that is almost identical in all organisms. Primers were obtained from DNA Technology, Aarhus, Denmark, and probes from TIB-MOLBIOL, Berlin, Germany. Probe and primer sequences were analysed by BIOBASE  and BLAST .Primers and probes were designed from the The PCR product was 144 bp in size, which according to the manual  is preferable to perform an efficient quantification of DNA.2 (PCR buffer), 2 μl of ready-to-use Fast Start DNA Master Hybridization Probes  (contains a hotstart Taq DNA polymerase and reaction mixture), 1.5 μl Uracil-DNA Glycosylase (heat-labile) and 2 μl of the DNA template. Water was added up to 20 μl, which was the final volume of all reaction-mix. For the DNeasy treated patient samples 4 μl of DNA was used. When proteinase K treated patient samples were examined (without DNeasy treatment), we used 2 μl of the undiluted DNA.Real-time PCR was performed in glass capillary tubes. The reaction mixture was composed of 0.5 μM of each primer, 0.2 μM of each probe, 5 mM of MgClM. hominis PG21 was added by use of filter pipette tips. Uracil-DNA Glycosylase  was used to prevent the samples from possible PCR "carry-over" contaminations from previous DNA synthesis reactions. Reaction mixes contained dUTPs instead of dTTPs, and therefore it was possible to avoid contamination of samples by adding an enzyme that hydrolyzes uracil-glycosidic bonds at U-DNA in single and double-strained DNA [To avoid contamination, mixing of the reagents (except of the DNA template) was performed in a separate room, away from rooms where culturing and DNA purification were done. The DNA template from ined DNA .M. hominis PG21 DNA. The concentrations of standard dilution series were: 105, 104, 103, 102, 101, 5 and 1 copy/μl. Siliconized tubes were used to prevent DNA from sticking to the wall of the plastic tubes. For carrier, yeast RNA was used in concentration 10 μg/ml.As negative control a sample containing all reagents except DNA was used in every PCR run. Quantification of DNA concentrations performed by LC-PCR Software was based on standard dilution series with known concentrations of genomic The LightCycler PCR program was composed by: Hotstart Taq DNA polymerase activation done in 95°C for 10 minutes, followed by cycling: 95°C (20°C/s) for 15 s, 58°C (20°C/s) for 8 s and 72°C (20°C/s) for 8 s, repeated 45 times. Melting assay ended the analysis: samples were heated to 95°C (20°C/s) without hold, cooled to 55°C (20°C/s) hold for 15 s and then heated slowly at 0.1°C/s up to 95°C, finally cooled to 40°C (20°C/s).Fluorescence emitted at 705 nm was measured at each annealing step since the fluorescence signal is emitted when both probes are hybridized. After annealing the temperature is raised and the hybridization probes are displaced by the Taq polymerase during the elongation step. Probe fluorescence was detected in canal F3 (measures at 705 nm) and F1 (measures at 530 nm). F3/F1 was used to correct differences in volume of the samples made during pipetting.Baseline Adjustment with the use of a "fit points" method, second step allows background reduction using Noise Band correction, and the last step is Analysis where the standard curve is generated from the threshold cycles (Ct) of the standard dilution series . Quantification software performs all additional steps for generation of a standard curve. First step involves ies fig. . SamplesNone declared.Author A.B. carried out the real-time PCR experiments, the analyses of data, and drafted the manuscript. Author H.F.S. participated in designing the LightCycler PCR method, analysis of data and coordination of the manuscript. Author J.F. participated in coordination of the study and provided clinical samples. Author S.B. participated in design and coordination of the study. Author G.C. participated in design, data analyses, coordination of the manuscript and study.


Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function.Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The We utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions.C. pneumoniae (Cp), C. caviae (GPIC), and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi.We show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of Caveolin-2 associates with the chlamydial inclusion independently of caveolin-1. The function of caveolin-2, either in the uninfected cell or in the chlamydial developmental cycle, remains to be elucidated. Nevertheless, this second caveolin protein can now be added to the small number of host proteins that are associated with the inclusions of this obligate intracellular pathogen. Chlamydiaceae are gram-negative obligate intracellular bacterial pathogens that replicate entirely within membrane bound inclusions that develop from the phagocytic vacuoles in which they enter. By mechanisms not understood in detail, inclusions avoid fusing with endosomes or lysosomes that might destroy the pathogen . In conThe fact that entry of some chlamydial strains is dependent on lipid rafts in general, rather than on the more specialized caveolae, was demonstrated using Fischer Rat Thyroid (FRT) cells, which do not express caveolin-1 or form caveolae. All ten strains that we examined were able to enter the caveolin-1 negative FRT cells, but not if those cells were treated with pharmacological agents that specifically disrupt lipid rafts .Chlamydia can interact.The Golgi is a likely source of most caveolin-1 on the inclusions of those strains entering via lipid rafts. This is suggested by the fact that caveolin-1 immunostaining of those inclusions actually increases as they enlarge and mature, and is consistent with reports that caveolin-1 first becomes associated with raft domains in the Golgi, and subsequently is transported to the plasma membrane on vesicles derived from the Golgi -23. MoreThe caveolin gene family also contains caveolin-2  which haC. trachomatis serovars A, B, and C. In the same cells, inclusions of C. trachomatis serovars E, F, and K, C. pneumoniae (A39), and C. caviae (GPIC) were marked by both of the caveolin proteins. Each of these eight strains acquired caveolin-2 even in caveolin-1 negative FRT cells. In addition, the ability of each of these strains to acquire caveolin-2 in the FRT cells was dependent on pathogen gene expression. Thus, chlamydiae appear to activate or induce an additional pathway for the transport of caveolin-2 that is independent of caveolin-1. Inclusions of two strains, LGV (L2) and MoPn, were not marked by either caveolin-1 or -2.In the present study, we ask whether caveolin-2, like caveolin-1, might be associated with inclusions formed by particular chlamydial strains. Unexpectedly, we found that in HeLa cells, caveolin-2 was associated with inclusions of strains that did not enter via lipid rafts and that did not acquire caveolin-1 either at entry or at later times. These strains included C. pneumoniae AR39 (Cpn), C. caviae, Guinea Pig Inclusion Conjunctivitis (GPIC strain), C. trachomatis serovars A/Har-13, Har-36B, C/TW-3, E/VW-KX, F, K/VR887, mouse pneumonitis agent, (MoPn) and Lymphogranuloma venereum, (LGV 434) were grown in HeLa 229 cells without centrifuge assistance. Infectious elementary bodies (EBs) were purified by renografin  density gradient centrifugation; alternatively lysates from infected cells were used to infect monolayers.HeLa 229 cells were obtained from the American Type Culture collection. Fischer rat thyroid (FRT) cells were a gift from Dr. Michael P. Lisanti . Cells were grown in minimum essential medium with insulin  with 5% fetal bovine serum .HeLa 229 or FRT cells were grown to confluence on 12 mm coverslips in 24 well plates . The cells were infected using the various chlamydial strains listed above at an MOI of 3.0 in complete cycloheximide overlay media  containing 10% FBS, 1X L-glutamine (CCOM) for 48 h at 37°C and 5% CO2. Coverslips with the cell monolayers were harvested, rinsed with phosphate buffered saline (PBS), fixed with 70% cold methanol, stored and subsequently immunostained following protocols similar to that described in detail previously ,19. BrieC. trachomatis serovars A, B, C, E, F, LGV, C. muridarum (MoPn), C. caviae (GPIC), and C. pneumoniae EB concentrates were exposed to short wavelength ultraviolet light for 1 h in a Biosafety hood. A portion of the EBs used was removed before UV treatment to serve as a control for initial viability. The EBs were then diluted in CCOM to an MOI of 4.0 per cell and used to infect HeLa 229 and FRT cells grown to confluence on 12 mm coverslips in 24 well plates. Plates were incubated for 2 h at 37°C and 5% CO2. After the 2 h incubation, the inoculum was removed and replaced with fresh CCOM; cells were then re-incubated for 34 h. At the end of the incubation period, the coverslips with cells were rinsed with sterile PBS and fixed with methanol for 10 minutes as detailed above. Following fixation, the cells were immunostained with a polyclonal guinea pig anti-chlamydia and mouse monoclonal anti-caveolin-2 antibodies as described above. FITC-conjugated goat anti-mouse and TRITC-conjugated goat anti-guinea pig secondary antibodies were used respectively to visualize the caveolin-2 and EBs within the cells. Slides were examined at 600X with a Laser Confocal Microscope system and images processed as described above.C. trachomatis serovar K and C. pneumoniae AR39 purified EBs were electrophoresed on a 4–12% NuPAGE gel  using MES running buffer. The separated proteins were transferred to a PVDF membrane at 25 V for 1 h using a 1X Novex® Tris Glycine buffer with 20% methanol in the XCell II™ Blot Module and a XCell Surelock™ Mini-Cell apparatus (Invitrogen). The blot was then rinsed with 0.1% BSA/PBS, blocked with 5% non-fat dry milk for 3 h, then rinsed and cut into two. One half was stained with a 1:200 dilution of mouse monoclonal anti-caveolin-2 antibody , while the second half was stained with a 1:100 dilution of a rabbit anti-Chlamydia for 2 h with shaking. The blot was then rinsed and a 1:1000 dilution of AP-conjugated goat anti-mouse secondary antibody added and incubated for 1 h at RT. Following several washes, a BCIP/NBT alkaline phosphatase substrate was added and the color allowed to develop.C. trachomatis serovars E, F, and K, C. pneumoniae (A39), and C. caviae (GPIC), previously were seen to enter HeLa cells via lipid rafts, and their inclusions were marked by caveolin-1 in those cells (ibid). Immunostaining with caveolin-2 specific monoclonal antibodies demonstrated that in HeLa cells, inclusions of each of these strains also were marked by caveolin-2 , and GPIC ; suggesting that the proteins might be co-localizing in hetero-oligomeric complexes.Chlamydia-containing vacuoles and later, the developing inclusions, are able to intercept and fuse with exocytic vesicles of the biosynthetic pathway that originate from the Golgi [Chlamydiae or not.It is well known that he Golgi ,7. Thesehe Golgi . In addihe Golgi ,26,30,31C. caviae) and C. pneumoniae (AR39) do not express IncG [To date, no chlamydial protein has been identified that might be secreted into the host cell cytosol to influence host intracellular trafficking. However, several chlamydial proteins, termed Incs, have been identified in the inclusion membrane -35. Seveess IncG , althougess IncG . There aChlamydia. As noted above, all ten of the chlamydial strains and species we examined were able to enter caveolin-1 negative FRT cells [C. trachomatis serovars A, B, and C, whether developing in HeLa or FRT cells, display only caveolin-2, suggesting a potentially broader chlamydial requirement for the caveolin-2 protein during development.Despite their presence on chlamydial inclusions, it remains unclear what role, if any, caveolin proteins may play in the developmental cycle of RT cells . Since tRT cells ,26, it wRT cells , the onlRT cells  (and curThe series of merged optical sections demonstrating the association of caveolin-2 with the inclusions (Figure Chlamydiae, depending on the serovar, or the species, one or both of the caveolin proteins may play an important role in the developmental cycles. Further studies of the interaction of the Chlamydiae with these enigmatic host cell proteins may well help to clarify caveolin functions in the host cell. Likewise, such studies may indicate factors influencing caveolin expression and trafficking from the Golgi, and elucidate their significance to the developmental cycle of these pathogens. The function of caveolin-2, either in the uninfected cell or in the chlamydial developmental cycle, remains to be elucidated. Nevertheless, this second caveolin protein can now be added to the small number of host proteins that are reported as associated with the inclusions of this obligate intracellular pathogen.Among the None Declared.ES and LN conceived of the study and participated in its design and coordination. LN was instrumental in the design of the study and prepared the initial draft of the manuscript. WW carried out all the experimental work, took photographs, did statistical analyses where necessary, and participated in the design and coordination of the project. All authors contributed to the preparation of the final manuscript.The pre-publication history for this paper can be accessed here:


At the interface of biology and nanotechnology lies an area of research that aims to construct molecular-scale machines based on protein and nucleic acid In 1959 Richard Feynman delivered what many consider the first lecture on nanotechnology. This lecture, presented to the American Physical Society at the California Institute of Technology, prompted intense discussion about the possibilities, or impossibilities, of manipulating materials at the molecular level. Although at the time of his presentation, the manipulation of single molecules and single atoms seemed improbable, if not impossible, Feynman challenged his audience to consider a new field of physics, one in which individual molecules and atoms would be manipulated and controlled at the molecular level .As an example of highly successful machines at the “small scale,” Feynman prompted his audience to consider the inherent properties of biological cells. He colorfully noted that although cells are “very tiny,” they are “very active, they manufacture various substances, they walk around, they wiggle, and they do all kinds of wonderful things on a very small scale” . Of courThe field of nanotechnology has indeed emerged and blossomed since Feynman's 1959 lecture, and scientists from many disciplines are now taking a careful look at the protein “machines” that power biological cells . These “As a graduate student in molecular biology, I have been especially interested in creative approaches to bridging the fields of biology and nanotechnology. Both DNA and protein molecules possess a number of intrinsic characteristics that make them excellent candidates for the assembly of dynamic nanostructures and nanodevices. Properties such as the site-specific molecular recognition among interacting protein molecules, the template-directed self assembly of complementary DNA strands, and the mechanical properties of certain protein complexes have enabled bionanotechnologists to envision a molecular world built “from the bottom up” using biological-based starting materials.In my own research, I have been very interested in investigating protein interactions and protein pathways on a genome-wide scale. In many ways, protein pathways are analogous to nanoscale “assembly lines,” since protein pathways often involve a series of proteins that act in successive order to yield a particular molecular “product” or perform a particular molecular function. While these protein-based “assembly lines” are commonplace within biological cells, they prompt two interesting questions with respect to the field of nanotechnology. First, can we mimic these multicomponent protein-based “assembly lines” on nanofabricated surfaces? And, second, can we tailor these “nanoscale assembly lines” to perform new and unique tasks?Nanomechanical protein complexes, such as the rotary ATP synthase complex, have also generated much interest from a nanotechnology standpoint . These pIn order to fully exploit these nanoscale protein machines, it is of prime importance to be able to position individual proteins and protein complexes at the nanoscale.Progress in this area has recently been reported by It is interesting to consider some of the applications of self-assembled protein arrays. 2 polypeptide forms a compact structure composed of two α-helices folded back onto each other. At low pH, HA2 undergoes a substantial conformational change, which results in a single “extended” helix. This conformational change results in a linear mechanical motion, with a linear movement of approximately 10 nm (In addition to rotary biomolecular motors, proteins that undergo substantial conformational changes in response to external stimuli might also find some interesting uses in nanoarrays. ly 10 nm . It woulThe work of As the field of nanotechnology continues to evolve, it is likely that we will see many more nanotechnology applications utilizing biological macromolecules. Toward the end of Richard Feynman's 1959 lecture, he quipped, “What are the possibilities of small but movable machines? They may or may not be useful, but they surely would be fun to make.”


G-protein inwardly rectifying potassium channel 1 (GIRK1) is thought to play a role in cell proliferation in cancer, and GIRK1 gene expression level may define a more aggressive phenotype. We detected GIRK1 expression in tissue specimens from patients with non-small cell lung cancers (NSCLCs) and assessed their clinical characteristics.Using reverse transcription-polymerase chain reaction (RT-PCR) analyses, we quantified the expression of GIRK1 in 72 patients with NSCLCs to investigate the relationship between GIRK1 expression and clinicopathologic factors and prognosis.In 72 NSCLC patients, 50 (69%) samples were evaluated as having high GIRK1 gene expression, and 22 (31%) were evaluated as having low GIRK1 gene expression. GIRK1 gene expression was significantly associated with lymph node metastasis, stage . The overall and stage I survival rates for patients with high GIRK1 gene expressed tumors was significantly worse than for those individuals whose tumors had low GIRK1 expression .These data indicate that GIRK1 may contribute to tumor progression and GIRK1 gene expression can serve as a useful prognostic marker in the overall and stage I NSCLCs. Lung cancer is one of the leading causes of cancer death in North America . Lung caG-protein inwardly rectifying potassium channels (GIRK) are found in both the heart and the brain, where they are associated with a slowing of the heart rate and suppression of neuronal response . The chaTumor tissue was collected from 72 patients with NSCLC who underwent curative surgery between 1993 and 1995 at Department of Surgery, Teikyo University School of Medicine. Patients who died within one month after surgery and patients with a past history of another cancer were excluded from the study. Of the 72 patients included, 52 were men and 20 were women and their ages ranged from 34 to 80 years (mean of 66 years). With regard to histological type, 41 were adenocarcinomas, 28 were squamous cell carcinomas and 3 were large cell lung carcinomas. The lesions of these 72 patients were staged on both operative and pathologic findings according to the UICC TNM classification (1997) [Total RNA was purified from fresh soft tissues by the acid guanidinium-thiocyanate procedure . The humThe densitometric value obtained for the GIRK1 band of a given tumor tissue sample was divided by the corresponding S14 value, and was referred to as the GIRK1 gene expression rate. The level of the GIRK1 mRNA expression in PC-14 cell line is elevated. The expression ratio of the tumor was then divided by that of the human pulmonary adenocarcinoma cell line PC-14 to obtain the GIRK1 conservation rate. When the conservation rate of a given specimen was ≧ 0.8, it was considered to indicate high expression of the GIRK1 gene, and if the rate was < 0.8, it was defined as low expression.ratio, it was considered to indicate high expression of the AMF-R gene, and if the rate was lower than the mean ratio, it was defined as low expression of the AMF-R gene.To ascertain whether links between GIRK1 gene expression and another gene expression, known as a marker of lymph node metastasis and tumor progression in NSCLC, we examined the relationships between GIRKI gene expression and AMF-R gene expression in 46 cases . The stuAll data regarding the clinical and histopathological variables were stored in a Macintosh computer. The Stat View program  was used for all statistical analyses. The relationship between the incidence of GIRK1 expression and clinico-pathologic factors, and AMF-R gene expression was examined by the chi-squared test with Fisher correlation. Survival curves were calculated using the Kaplan-Meier method and analyzed by the log-rank test. Statistical significance was identified as p < 0.05.To determine the number of PCR cycles appropriate for quantification, from 20 and 50 cycles of PCR were performed, in 5-cycle increments. The expression ratios of GIRK1 to S14 were reasonably constant 35 to 45 cycles (data not shown). Therefore, in the subsequent experiments the values obtained at 40 cycles were defined as the expression of the target genes. Using forty RT-PCR cycles, we found that the ratio of GIRK1/cell line PC-14 expression ranged from 0 to 2.2  in tumor specimens  were classified as AMF-R high gene expression, and 13(28%) were classified as having AMF-R low gene expression. In most GIRK1 high gene expression cases, AMF-R gene was also expressed. As shown Table % were clThe survival was compared between the high GIRK1 gene expression group and the low GIRK1 gene expression group in overall, stage I and stage II/III. Figures In spite of significant advances in surgery and the use of new, more effective chemotherapeutic regimens, the overall 5-year survival of patients with NSCLC is 17% . IdentifHuman carcinomas exhibit hyperpolarized membrane potential as compared with surrounding normal tissue ,15. GIRKIn this study, we used human pulmonary adenocarcinoma cell line PC-14 as a positive control for GIRK1 gene expression. The patients were classified into two groups according to the cutoff point of mean ration of GIRK1/cell line PC-14 expression in tumor specimens. The mean number has been widely used as the cutoff point to divide the patients into two groups ,21. GIRKIn conclusion, the present study suggests GIRK1 may be contribute to tumor progression and could be a useful prognostic marker in patients with overall and stage I NSCLC. Thus the current findings provide evidence to support a potential utility of this gene in developing a diagnostic test for NSCLC patients.The author(s) declare that they have no competing interest.IT: Data analysis and writing of manuscript. YI, MG: Critical appraisal of manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In sub-Saharan Africa, HIV/AIDS and violent conflict interact to shape population health and development in dramatic ways. HIV/AIDS can create conditions conducive to conflict. Conflict can affect the epidemiology of HIV/AIDS. Conflict is generally understood to accelerate HIV transmission, but this view is simplistic and disregards complex interrelationships between factors that can inhibit and accelerate the spread of HIV in conflict and post conflict settings, respectively. This paper provides a framework for understanding these factors and discusses their implications for policy formulation and program planning in conflict-affected settings. Of the obstacles to development in the sub-Saharan African (SSA) region, perhaps none has had a more profound impact than the dual burdens of HIV/AIDS and conflict. During the past quarter century, no region of the world has been more acutely affected by large-scale violent conflict than SSA. Almost all SSA countries have directly experienced, or border a country that has directly experienced, violent conflict. The number of states engaged in significant violent conflicts doubled between 1989 and 2000, from 11 to 22 [The same quarter century has seen the development of an even more pervasive crisis – the HIV/AIDS pandemic. SSA continues to account for the large majority (66%) of the world's HIV/AIDS cases , even thAlthough much has been written about HIV and conflict individually, surprisingly little has been written about the dynamics of the relationship between the two crises. In fact, an extensive keyword search of the internet and peer-reviewed journal databases turned up fewer than 100 references to the intersection of HIV and conflict. Virtually all studies are descriptive and only one utilized hypothesis-testing analytical strategies . AlthougSome literature on conflict-affected countries in SSA underscores mechanisms by which HIV/AIDS and violent conflict may exhibit bi-directional causal associations on the population level. HIV/AIDS has been recognized to play a potential role in creating conditions conducive to violent conflict on the continent, although there is as yet no good empirical evidence to that effect. A policy forum held at the United States Institute for Peace  concludeOn the other hand, violent conflict clearly influences the epidemiology of HIV. To date, the literature emphasizes conflict as a risk factor for HIV transmission. Conflict destroys social and physical infrastructure, resulting in untreated sexually transmitted infections (STIs), poor health and malnutrition and, as a consequence, increased risk of transmission in the event of viral exposure. The migration and poverty created or exacerbated by conflict may result in increased exposure opportunity through: (1) increased prevalence of casual or commercial sexual activity; (2) increased interactions among civilians and combatants/military personnel, known for their high risk behaviors; (3) the development of cultures of violence that promote sexual violence and predation; (4) mass migration, which increases sexual mixing among populations; and (5) the destruction of public health education mechanisms , which negatively affects public health-related knowledge, attitudes, and practices ,9.2, suggest that the relationship between conflict and HIV levels may be more complex than this general picture of joint potentiation implies. With few exceptions, countries that have experienced widespread violent conflict have apparently lower levels of HIV infection compared to those that have experienced relative peace , which ultimately affects disassortive mixing . In Southern Africa, the regional ecology is one that favors extraordinarily high HIV risk through, for example, high population density, good physical infrastructure, economically-motivated mobility, high poverty levels, and large wealth disparities within and between countries. By lowering exposure opportunity, war may lead to isolation from the general regional ecology and, therefore, to lower HIV risk at the population level. Alternatively, war leads to a changed and often more intense mixing of mobile military populations and civilians, which can increase HIV risk through disassortive mixing.or decreased during conflict. The relative importance of each of the components, and therefore the ultimate effect of conflict (i.e. augmenting or reducing risk), will be highly context specific.Figure Figure This finding is critical in that it explains in part why, in some chronic conflict settings, apathy has developed towards HIV because seroprevalence levels may be low. However, the potential for rapid progression of the infection post conflict due to high vulnerability argues for a much more aggressive and deliberate approach to HIV in post-conflict settings.determinants of conflict or the manifestations of conflict. Typologies that focus on the determinants of conflict categorize conflicts primarily based upon characteristics of the political, legal, economic, social, or cultural environments that are associated with greater risk of conflict. Examples of such factors include [Considerable research has been conducted to date to construct comprehensive typologies of conflict. Several well-established research initiatives, such as the Correlates of War Project , have de include :• political/legal status and structure of warring parties ;• economic context ;• nature of political system (e.g. democratic or authoritarian);• self-identified characteristics distinguishing warring parties ;• characteristics of physical environment as it affects the conflict (e.g. mountainous or flat); and• purpose of conflict .In contrast, some approaches focus on the manifestations of conflict, defining categories in terms of measurable aspects of the conflict and its effects on populations. Examples include:• geographic scale of conflict ;• time-scale of conflict (e.g. duration and onset);• types of military technology, tactics, and funding methods employed;• involvement of civilians;• levels of mortality (e.g. battle-related and indirect);• levels of other negative health outcomes ; and• levels of displacement .A determinant-based typology has more direct application to the critical areas of conflict early warning and prevention, despite the analytical complexities derived from the high degree of interdependence (i.e. interaction) among these determinants . HoweverDespite large cross-cultural and historical variation in the determinants and manifestations of conflict, and variation in the systems that have been developed to analyze conflict, a model can be developed specific to inquiry about the public health impacts of conflict on HIV epidemiology. Conflict can be analyzed in terms of the following dimensions, all of which may help to shape its influence on HIV risk.duration of the conflict and the point of conflict onset relative to the progression of the epidemic should be considered. Chronic conflict, for example, may engender profound societal changes that are manifested in immediate increases in population risk of HIV. For example, widespread impoverishment resulting from war may fuel high levels of commercial sex as a survival strategy. Similarly, displaced populations are believed to be at greater risk of sexual violence or sexual activity as a way to negotiate access to key resources for survival. A recent finding regarding the behavior of humanitarian aid workers in western Africa tragically supports this assertion [Both the ssertion .Longer-duration conflicts generally lead to greater cumulative effects of conflict on social infrastructure. Populations may be isolated from modern communications for a long period of time, resulting in much greater naïveté regarding the epidemiology and prevention of the disease. In low-prevalence conflict settings this may not impact transmission significantly; post conflict, however, this may exacerbate vulnerability.Alternatively, chronic conflict may result in lower exposure opportunities following reduced social mixing due to isolation and limited population mobility. During the war in Angola, for example, mobility was limited and most of the population was concentrated in small "islands of security" around provincial capitals. HIV seroprevalence remained low as a result.Equally, the point of conflict onset relative to the progression of the epidemic may determine the degree to which conflict may result in sustained low seroprevalence levels for the duration of the war. In Rwanda, for example, sentinel surveys among pregnant women and STI patients demonstrated prevalence rates exceeding 30% before the 1994 genocide . Becausepolitical/legal status, differential HIV prevalence rates, relative size, motives, and tactics characterizing combatants and affected civilian populations may all shape the progression of the epidemic. In terms of political/legal status, combatant groups may comprise government military forces, paramilitary forces, organized rebel groups, or highly fragmented rebel groups. Available data suggest but do not conclusively confirm the oft cited axiom that significant differentials in seroprevalence can exist between government military and civilian populations in Africa [The n Africa . More ren Africa . Almost Modern conflict in SSA often inflicts a high degree of sexual violence upon civilian populations that, if civil-military seroprevalence differentials exist, will result in increased mixing and HIV spread. Additionally, commercial sexual activity and other commercial activities may follow soldiers, again facilitating HIV spread. The size and motives/tactics of armed groups may determine in part the types of interactions (and violence of those interactions) with the civilian population.scale and geographic focus of the conflict highlight geographic areas that may be differentially affected by the conflict or the epidemic. In terms of scale, conflicts may take place on a regional, national, or sub-national level. Most post-Cold War conflicts in SSA involve civil conflict; many conflicts have international and regional dimensions. The war that started in the Democratic Republic of Congo (DRC) in 1998 drew armies from at least seven African countries experiencing wide variations in their levels of HIV prevalence. The concept of the geographic focus of the conflict highlights the fact that the effects of war are unlikely to be homogeneous in any situation. This gives rise to distinct local ecologies that may experience lower HIV vulnerability and exposure to HIV hazards through the factors listed above, particularly isolation and population mixing associated with military and forced migration. For example, the Ethiopian conflict resulted in a large concentration of military along the Ethiopian-Eritrean border and accompanying civilian commercial activity. In some cases, such as Mozambique, conflicts have resulted in intensified and highly localized trading corridors between outside countries of higher seroprevalence and lower prevalence areas.The Factors may decrease risk by decreasing vulnerability, or more commonly, by decreasing exposure opportunity. The following factors are postulated to accompany conflict in post-Cold War SSA and may explain how conflict may actually limit the spread of the epidemic during the period of conflict.Conflict isolates communities by destroying transport systems, making travel unsafe, and disrupting the market-based activities that encourage economic migration. Conflict may freeze normal cross-border migration, as in the case of Ethiopia and Eritrea. On the sub-national level, civil conflict may isolate rural communities controlled by armed factions, as in Angola, or limit population movement because of the loss of transport infrastructure, as in the Democratic Republic of Congo. Where conflict is chronic, the effect of limiting population movement and therefore population mixing could be a very significant contributor to the relatively lower levels of HIV found among affected populations.Another aspect of conflict that may decrease population HIV risk is differential mortality among high-risk groups. It is well established that mortality among adult males is elevated in conflict-affected settings. Post-genocide Rwanda has shown a demographic shift such that among adults between the ages of 15 and 54 years, women far outnumber men . In addi(guhahamuka); and the prevalence of a local, less acute syndrome of "severe grief" (agahinda gakabije). In the commune that experienced significantly lower levels of violence during the genocide (Butamwa), only 5.6% of adults had depression, and 31.9% of adults had agahinda gakabije. In the commune that experienced widespread violent conflict during the genocide, almost one-fifth (17.9%) of adults had depression and almost half (41.8%) of adults had agahinda gakabije [Available evidence points to the unexplored possibility that conflict may result in decreased sexual activity, most commonly as a result of psychological sequelae of trauma such as post-traumatic stress disorder (PTSD). It has been shown that absent or low libido in those with post-traumatic stress disorder can be as high as 69% . In thosgakabije . The resConflict may lead to the disruption of sexual networks associated with forced migration and/or the conscription of husbands and sexual partners. Females may have fewer opportunities for casual sex, though this factor remains unexplored in the research literature. This may hold most true in displaced communities, which are often comprised disproportionately of women and children.Accelerating factors in this model constitute factors that tend to enhance the spread of HIV by worsening dimensions of population vulnerability or increasing HIV exposure opportunity.Conflict has been demonstrated to result in the increased sexual mixing of military with civilian groups, especially in areas of high military concentration for extended periods of time . In postImpoverishment coupled with discrimination against women and the erosion of traditional behavioral norms may give rise to high levels of sex driven by economic motives in conflict and transitional societies. This again is frequently cited as an important consequence of war, but is difficult to quantify. What is clear is that African countries that have experienced war have lasting socioeconomic effects. For example, Mozambique and Angola are among the three lowest ranking countries on all components of the Human Development Index among Southern African countries . Infant Destruction of the health sector is a common feature of conflicts and its reconstruction has not been rapid in most SSA countries. Massive loss of health infrastructure – both personnel and physical infrastructure – is common in conflict-affected countries of Africa. Mozambique lost the majority of its clinics during the civil war . Rwanda Not only is infrastructure lost, but utilization patterns also are affected by violent conflict. People may not be able to get to health care services because of ambient danger. They may also be reluctant to use services because they distrust providers. This may have been a factor in Rwanda, for example, where providers were particularly implicated in genocide activities during the war . AnotherWhile quality evidence-based research demonstrating direct links between malnutrition and increased susceptibility to HIV seroconversion is scarce, the importance nutrition plays in limiting infection and regulating the immune system is well documented. Malnutrition and micronutrient deficiencies impair immune function, thereby increasing vulnerability to infections in general. Vitamin A, for example, has been shown to play an important role in immune function, including the maintenance of mucosal epithelia, the growth of immunogenic cells and antibody response . SupplemIn addition, malnutrition levels are generally a good indicator of health status and social equity. High levels of micronutrient deficiency and general malnutrition are widely documented in conflict-affected populations, resulting in decreased resilience to infections. It is known that poor nutritional status is associated with vulnerability to progression from tuberculosis infection to disease. The immunosuppression associated with HIV infection is a major risk factor for the progression of latent tuberculosis infection to active disease and death . The incThe isolation and poverty caused by war often results in decreased knowledge of, and access to, means to prevent HIV transmission. Population probability surveys available through the Demographic and Health Surveys, UNICEF's Multiple Indicator Surveys, and specialized HIV behavioral surveys show that knowledge levels and condom use are quite low in conflict-affected countries  and thatAdditionally, knowledge levels tend to exhibit marked intra-national variability, particularly among socially and economically marginalized groups. This variability may be enhanced in conflict-affected countries through regional isolation .The case of Rwanda suggests that conflict may affect contraceptive use and desired family size. Popular wisdom suggests that post-conflict populations may want to repopulate and, to an extent, that tendency is supported by examination of Demographic and Health Survey (DHS) data collected before and after the genocide of 1994. In 1992 and 2000, the DHS documented rates of awareness of HIV exceeding 80% and awareness of modern methods of contraception exceeding 90% . DespiteOn the whole, forced migration may increase HIV risk, as forced migration movement tends to be in a rural-to-urban direction, which greatly enhances the possibility of disassortive mixing. In addition, the migration process is often associated with high levels of physical danger and exposure to sexual violence. Where forced migrants remain in highly insecure areas, migration may be associated with frequent assaults by combatants. In addition, migration patterns may be quite fluid, enabling dislocated populations to move back and forth between higher and lower risk areas, thus increasing disassortive mixing. Research suggests that urban rates of casual/commercial sexual activity tend to exceed rural rates and that exchange of rural/urban populations tends to undermine traditional norms governing sexual activity in rural areas . It has Fluid population movements are particularly common during prolonged conflicts in border areas that might give rise to population mixing and then repatriation. In the case of Rwanda, research illustrates that the post-war period has seen a sharp decrease in the HIV prevalence differential between rural and urban women attending antenatal clinic services in Kigali . The treCoupled with increased population movement is the frequent emergence of norms of sexual predation and sexual violence within conflict-affected areas. The phenomenon of rape as a war tactic is increasingly being recognized and documented. The widespread infliction of sexual violence upon women during the Rwandan genocide and its aftermath illustrates the extent to which sexual violence may be utilized as an instrument of war. While exact figures are unavailable, it is estimated that between 250,000 and 500,000 cases of rape occurred during the conflict ,62. In LIn cases of HIV resulting from sexual violence, the shame and social stigma attached to rape prevent women from seeking testing or care. It should be noted that in some conflict-affected populations, research suggests discriminatory cultural attitudes and practices. In Rwanda, research in 1990 found that men control sexual decision-making, and that HIV positive women were more likely to report coercive sex and violence with their sexual partner than HIV negative women . In AngoLike HIV, conflict decimates family structures through mortality and dislocation, and results in lasting effects on society . ConflicThe models and mechanisms identified above help to develop an explanatory model built on a foundation of social ecology. A social ecology approach argues for models of explanation that explore the interactions of multiple causes  working through different mechanisms and at differing levels of scale  with emphasis on dynamic change over time. The social ecology of the epidemic is defined in terms of sociodemographic, socioeconomic, structural, contextual, biologic and behavioral variables that facilitate or inhibit progression of the epidemic -70. The One key feature of mapped data is the relative correspondence of population density, road infrastructure, economic productivity, and HIV risk in Southern Africa . Population density paired with excellent road infrastructure may have a synergistic effect on HIV risk. This may, in part, explain the pattern of risk seen in southern and eastern Africa.Economics and poverty are important drivers. Although higher economic output is associated with higher levels of HIV in general, the poor are increasingly affected for a variety of well-accepted reasons. From this perspective, Southern Africa remains the most problematic with respect to the potential for rapid northward spread of HIV. Angola and Mozambique are characterized by high poverty, economic potential, and in the case of Angola, the potential to be a gateway to West Africa. One factor that may be driving the epidemic is the interaction between poor women and rich men  as exemplified by Drain et al.  who founThe ecology of HIV and conflict also results in greater micro-level variation in the determinants of HIV risk. Mock and Drapcho  showed tSadly, the international community has compartmentalized its responses to HIV and conflict – as well as its relief, recovery, and development programming instruments – which precludes an integrated and aggressive attack on the epidemic. As a result, very little has been learned in terms of designing and implementing programs to address HIV among conflicted-affected populations, with some exceptions .The current structure of development assistance results in poor availability of resources to address HIV in conflict settings, as these are traditionally the domain of humanitarian assistance. To date, though much lip service is paid to the concept of developmental relief, the bulk of all humanitarian assistance is supply-side delivery of immediate survival commodities and services. This approach continues to dominate even though humanitarian assistance may be provided to conflict-affected population for years and decades, and even when displacement may provide unique opportunities to reach populations that might otherwise be inaccessible.Similarly, the immediate post-conflict period is typically serviced by transition programs that emphasize demobilization, reinsertion, reintegration and development of the foundations of governance. While these are clearly important considerations, a problem as severe as HIV cannot be compartmentalized as a development problem (i.e. not a humanitarian or transition-period concern). And indeed, problem-focused activities such as HIV prevention might be a motivating cause for the stimulation of civil society groups.Even internal to the development community, the problem of HIV is not linked to the problem of conflict, but rather these two issues are seen and treated as unrelated concerns. Different strategies and partners are managed by separate bureaucratic units that have rare interactions. Such compartmentalization of both conflict and HIV along humanitarian/development lines and internal to development management has resulted in fragmented approaches to addresses these important and interactive influences on the health development of SSA.Similarly, the transitional period should be assessed for opportunities to creatively integrate HIV-related interventions into priority program strategies to promote economic recovery and social rehabilitation. Examples of such integrated programs include integrating HIV education into micro-credit or economic development programs and incorporating HIV risk reduction strategies into demobilization and social integration programs. Demobilized ex-combatants might also be incorporated in educational and health extension programs such that they become part of the solution instead of the problem.Humanitarian assistance provides numerous opportunities for the cross-sectoral integration of effective HIV-directed initiatives, including the opportunity for synergistic amelioration of adverse health outcomes. Even though conflicts typically last for years or decades, countries affected by conflict continue to receive assistance, primarily for immediate survival needs, through humanitarian assistance efforts. In post-accord transition programs, the emphasis is on establishing democratic rule, governance, and re-integration. Sequential application of traditional humanitarian and transition interventions ignores the regional context of high HIV risk. Since HIV risk may be lower in conflict settings while vulnerability to HIV is increasing, this programming approach results in a major constraint to timely, prevention-oriented intervention.Our analysis suggests that the relationship between conflict and HIV is complex and contextualized; however, important general conclusions can serve as a basis for action. The analysis demonstrates that while vulnerability to HIV is heightened as a result of conflict, exposure opportunities may be significantly reduced. This may lead to important opportunities for interventions to keep HIV prevalence rates low in the affected settings . More urgently, post-conflict changes in exposure opportunities could result in explosive epidemic waves. The case of Angola is particularly troublesome given its strategic location and economic potential.Conflict and HIV are inter-related problems that demand a clear strategy and coordinated use of humanitarian and development assets. This means that objectives for addressing these problems should drive the response, and conflict should be viewed as a key determinant of HIV risk. The HIV/AIDS epidemic is a major constraint to development in Africa. Programs and bureaucracies should more clearly align along a consistent vision of HIV prevention and mitigation in both conflict and post-conflict settings.A portion of the Declaration of Commitment on HIV/AIDS, adopted at the United Nations General Assembly Special Session on HIV/AIDS (UNGASS) on June 27, 2001, articulates strategies and goals to address HIV/AIDS in conflict and disaster-affected regions. It calls for the development and implementation of national strategies that incorporate HIV/AIDS awareness, prevention, care, and treatment elements into programs or actions that respond to emergency situations. The declaration recognizes that populations destabilized by armed conflict, humanitarian emergencies, and natural disasters – including refugees, internally displaced persons and, in particular, women and children – are at increased risk of exposure to HIV infection, and calls for HIV/AIDS components to be factored into international assistance programs where appropriate .As international, regional and national agencies strive to abide by the Declaration of Commitment on HIV/AIDS, the framework provided here will be useful in the identification of determinants of HIV risk. We recommend that factors affecting HIV risk in conflict settings be systematically assessed as a basis for strategic planning to address HIV in conflict. The assessment should include vulnerability profiling, exposure risk assessment, and characterization of mechanisms through which conflict affects HIV risk. It is particularly important to pre-empt disassortive mixing associated with post-conflict improvements in mobility and resettlement. The assessment should be conducted at the different planning levels .Conflict risk assessment should also be a key component so that synergistic programming between conflict and HIV initiatives can be achieved. Examples of these include:• potentiating civil society organizations around the HIV problem, especially in areas of high prevalence;• incorporating HIV prevention as a key element in demobilization and reinsertion initiatives, including the possible use of ex-combatants as HIV educators/mobilizers; and• aggressive and progressive approaches to poverty alleviation and reduction. Poverty is particularly problematic in chronic conflict contexts, even when societies have high development potential (e.g. the DRC and Angola). Aggressive development programs are probably the most central strategy to confronting HIV and conflict. HIV prevention and conflict resolution can be inserted into a number of the specific components of these programs. Targeting women- and child-headed households, and making it economically feasible for families to send youth to school are also of particular importance.Our analysis stresses the importance of maintaining a global perspective, while at the same time recognizing that micro-planning may be even more important in conflicted-affected settings than in stable settings. This is because local ecologies may be more diverse due to the varying spatial and temporal effects of conflicts. Indeed, most countries that have experienced large-scale conflict have had quiet zones where life was relatively stable, even during the course of large-scale war. These stable areas could absorb major non-emergency initiatives. In post-conflict settings, these micro-level differences persist, while others, such as differing levels of infrastructure destruction, ethnic tensions and pockets of high HIV prevalence, may require highly specific approaches.These findings also suggest somewhat different approaches to HIV surveillance in conflict-affected settings. First, a more deliberate attempt should be made to support surveillance in countries and areas affected by conflict so that a better evidence base is developed. Unfortunately, a review of the UNAIDS surveillance database revealed that conflict-affected countries have little or no systematic surveillance, despite the existence of relatively stable areas . This maFinally, we argue that information strategies, including HIV/conflict risk assessments and surveillance, should be rationally planned and implemented as a basis for intervention planning and program evaluation during the early phases of conflict response. Seroprevalence or other proxy measures of HIV infection must be monitored more deliberately as a part of surveillance. Conflict-HIV vulnerability/risk assessment tools can be developed based on the factors enumerated above. They should be applied periodically to more effectively respond to a dynamic setting. Improved HIV status information together with these planning data should foster a more evidence-based approach to preventing and mitigating HIV in conflict settings. We call for mainstreaming HIV/AIDS prevention and care policies into conflict prevention, peacekeeping operations, humanitarian responses to crises, post conflict reconstruction planning, implementation and evaluation.The author(s) declare that they have no competing interests.Dr. Mock responsible for overall framework and approach for this paper. Drs. Duale and Brown contributed HIV literature synthesis. Ms. Mathys, O'Maonaigh, Abul-Husn and Elliot extracted literature. All authors read and approved the manuscript.


Bony defects arising from tumor resection or debridement after infection, non-union or trauma present a challenging problem to orthopedic surgeons, as well as patients due to compliance issues. Current treatment options are time intensive, require more than one operation and are associated with high rate of complications. For this reason, we developed a new surgical procedure to bridge a massive long bone defect.in situ periosteal sleeve is elevated circumferentially off of healthy diaphyseal bone adjacent to the bone defect. Then, the adjacent bone is osteotomized and the transport segment is moved along an intramedullary nail, out of the periosteal sleeve and into the original diaphyseal defect, where it is docked. Vascularity is maintained through retention of the soft tissue attachments to the in situ periosteal sleeve. In addition, periosteal osteogenesis can be augmented through utilization of cancellous bone graft or in situ cortical bone adherent to the periosteal sleeve.To bridge the gap, an The proposed procedure is novel in that it exploits the osteogenic potential of the periosteum by replacing the defect arising from resection of tissue out of a pathological area with a defect in a healthy area of tissue, through transport of the adjacent bone segment. Furthermore, the proposed procedure has several advantages over the current standard of care including ease of implementation, rapid patient mobilization, and no need for specialized implants  or costly orthobiologics.e.g. the Ilizarov procedure) as well as areas of the world where surgeons have little access to expensive, complex devices and orthobiologics.The proposed procedure offers a viable and potentially preferable alternative to the current standard treatment modalities, particularly in areas of the world where few surgeons are trained for procedures such as distraction osteogenesis ( Surgical treatment modalities involving auto/allografting and bone regeneration via distraction osteogenesis are complex, time intensive procedures of inherently high risk due to vagaries of organ donation, in the case of allografts, and the complexity of soft and hard tissue salvage during the process of distraction osteogenesis. In addition, orthobiologics are costly and their dosage regimes as well as efficacy are currently the subject of much research [Replacement of bone where there is none is one of the most challenging problems facing orthopedic surgeons today. In the case of tumor resection or trauma, massive bone defects must be filled with regenerate bone as quickly as possible in order to restore function. Current standards for bridging of massive bone defects in long bones generally follow a theme of research -3.Both bone grafting and bone transport procedures are complex for the surgeon as well as for the patient. Furthermore, they are susceptible to complications such as delayed union, extensive treatment time periods, infections, and insufficient mechanical function outcomes that can result in fractures. The high complication rates of these procedures exacerbate the previously mentioned difficulties associated with these treatment modalities, from the perspective of the surgeon as well as that of the patient. In short, the inherent risk of complications increases the need for patient compliance and clinical follow-up. Despite the effort associated with these procedures, their results are often less than satisfactory. Hence, the complexity and shortcomings of current state-of-the-art surgical procedures have provided impetus to develop a new treatment modality that provides a relatively straightforward, single step procedure with a high probability of success for the bridging of massive bone defects in long bones. The procedure is straightforward and can be implemented in operating rooms across the world without the need for high-tech equipment or expensive orthobiologics. The purpose of this manuscript is to describe the novel procedure.The proposed procedure is applicable for clinical scenarios including tumor resection as well as debridement after an infection or non-union. In the case of reconstruction after tumor resection Fig. , a transThe proposed procedure depends to a large degree on bone's inherent healing strategies. Bone is a remarkably resilient tissue capable of adaptation to the most extreme biological and mechanical environments; this capacity for self-regeneration without scarring is based on bone's endogenous healing strategies. First, bone remodels itself through osteoclastic resorption and osteoblastic matrix apposition; by constantly reweaving itself, the structure is dynamic and optimal for prevailing mechanical function. Furthermore, the natural healing cascade of bone after trauma recapitulates embryonic endochondral ossification. Hence, modeling, growth and remodeling confer a means to regenerate functional tissue at any time in the life cycle of a bone. The "raw materials" necessary to replace bone are located in the environment or produced by the cells that do the work of regeneration, i.e. osteoclasts and osteoblasts. In the case of regeneration of bone in defects, further potentially key constituents to the formation of a functional regenerate in situ include a patent blood supply, chemical gradients of morphogens and/or cytokines, a template onto which the cells can anchor themselves during the rebuilding process (e.g. graft or a scaffold), and biophysical stimuli such as fluid flow and/or cell level strains. The proposed procedure essentially replaces the defect site in a pathological zone with a defect site in a healthy bed of tissue and provides for progenitor cells through the surrounding, healthy periosteum as well as many of the other key constituents for successful tissue regeneration, as defined above.proof of feasibility for the proposed procedure. An 11-year old male presented with a low grade surface osteosarcoma of the tibia. After resection of the tumor, the fibula was resected for transfer and the surrounding periosteum was left behind to serve as an osteo-inductive and -conductive sleeve  as well as where surgeons have less access to expensive, complex devices and orthobiologics.The proposed procedure is novel in that it introduces for the first time the possibility to bridge a massive defect in a long bone using a single stage procedure. Furthermore, the proposed procedure has several advantages over the current standard of care including ease of implementation, lack of requirement for specialized implants  or costly orthobiologics, and rapid patient mobilization. This makes the proposed procedure a viable and potentially preferable alternative to the current standard treatment modalities, particularly areas of the world where few surgeons are trained for procedures such as distraction osteogenesis  declare that they have no competing interests.UK conceived of the technical innovation, organized its design and coordination, and drafted the manuscript. DSS was the senior surgeon in the clinical case showing feasibility. Both authors read and approved the final manuscript.