The means by which vaginal microbiomes help prevent urogenital diseases in women and maintain health are poorly understood. To gain insight into this, the vaginal bacterial communities of 396 asymptomatic North American women who represented four ethnic groups (white, black, Hispanic, and Asian) were sampled and the species composition characterized by pyrosequencing of barcoded 16S rRNA genes. The communities clustered into five groups: four were dominated by Lactobacillus iners, L. crispatus, L. gasseri, or L. jensenii, whereas the fifth had lower proportions of lactic acid bacteria and higher proportions of strictly anaerobic organisms, indicating that a potential key ecological function, the production of lactic acid, seems to be conserved in all communities. The proportions of each community group varied among the four ethnic groups, and these differences were statistically significant [χ 2 (10) = 36.8, P < 0.0001]. Moreover, the vaginal pH of women in different ethnic groups also differed and was higher in Hispanic (pH 5.0 ± 0.59) and black (pH 4.7 ± 1.04) women as compared with Asian (pH 4.4 ± 0.59) and white (pH 4.2 ± 0.3) women. Phylotypes with correlated relative abundances were found in all communities, and these patterns were associated with either high or low Nugent scores, which are used as a factor for the diagnosis of bacterial vaginosis. The inherent differences within and between women in different ethnic groups strongly argues for a more refined definition of the kinds of bacterial communities normally found in healthy women and the need to appreciate differences between individuals so they can be taken into account in risk assessment and disease diagnosis. T he human body harbors microorganisms that inhabit surfaces and cavities exposed or connected to the external environment. Each body site includes ecological communities of microbial species that exist in a mutualistic relationship with the host. The kinds of organisms present are highly dependent on the prevailing environmental conditions and host factors and hence vary from site to site. Moreover, they vary between individuals and over time (1). The human vaginal microbiota seem to play a key role in preventing a number of urogenital diseases, such as bacterial vaginosis, yeast infections, sexually transmitted infections, urinary tract infections (2-9), and HIV infection (10, 11). Common wisdom attributes this to lactic acid-producing bacteria, mainly Lactobacillus sp., that commonly inhabit the vagina. These species are thought to play key protective roles by lowering the environmental pH through lactic acid production (12, 13), by producing various bacteriostatic and bacteriocidal compounds, or through competitive exclusion (13-16). The advent of culture-independent molecular approaches based on the cloning and sequencing of 16S rRNA genes has furthered our understanding of the vaginal microbiota by identifying taxa that had not been cultured (17-24). However, this technique is limited by high cost and low throughput, hence only small ...

Heart muscle is characterized by a regular array of proteins and structures that form a repeating functional unit identified as the sarcomere. This regular structure enables tight coupling between electrical activity and Ca 2؉ signaling. In heart failure, multiple cellular defects develop, including reduced contractility, altered Ca 2؉ signaling, and arrhythmias; however, the underlying causes of these defects are not well understood. Here, in ventricular myocytes from spontaneously hypertensive rats that develop heart failure, we identify fundamental changes in Ca 2؉ signaling that are related to restructuring of the spatial organization of the cells. Myocytes display both a reduced ability to trigger sarcoplasmic reticulum Ca 2؉ release and increased spatial dispersion of the transverse tubules (TTs). Remodeled TTs in cells from failing hearts no longer exist in the regularly organized structures found in normal heart cells, instead moving within the sarcomere away from the Z-line structures and leaving behind the sarcoplasmic reticulum Ca 2؉ release channels, the ryanodine receptors (RyRs). These orphaned RyRs appear to be responsible for the dyssynchronous Ca 2؉ sparks that have been linked to blunted contractility and, probably, Ca 2؉ -dependent arrhythmias in diverse models of heart failure. We conclude that the increased spatial dispersion of the TTs and orphaned RyRs lead to the loss of local control and Ca 2؉ instability in heart failure.calcium signaling ͉ local control ͉ dyssynchrony ͉ heart failure ͉ transverse tubules C a 2ϩ sparks, the elementary Ca 2ϩ release events during the excitation-contraction (EC) coupling process in heart (1-5), are triggered by the opening of L-type Ca 2ϩ channels (LCCs) and sum to produce the [Ca 2ϩ ] i transient (6). Each Ca 2ϩ spark arises from a nanometer-sized structural and functional unit or ''couplon,'' which includes both (i) LCCs in the sarcolemma or transverse tubules (TTs), and (ii) a cluster of ryanodine receptors (RyRs) that reside in the sarcoplasmic reticulum (SR) membrane and face the LCCs across a 15-nm gap (7-9). During the cardiac action potential (AP), the nearly simultaneous opening of LCCs synchronizes the triggering of Ca 2ϩ sparks from the RyR clusters (also referred to as Ca 2ϩ release units, or CRUs). Ca 2ϩ signaling in heart failure is characterized by contractile dysfunction (10, 11) (24). In tachycardia-induced HF, Kamp and colleagues (25, 26) have described a regional loss of TTs within ventricular myocytes, but other evidence suggests that TTs remain normal. † † Additionally, many other factors may contribute to arrhythmogenesis, including reduced K ϩ channel expression, increased Na ϩ ͞Ca 2ϩ exchanger activity (21, 27), and altered Ca 2ϩ channel gating (28); therefore, possible links between structural changes and dysfunctional Ca 2ϩ signaling in this HF model remain unclear. In a cell culture investigation using pig ventricular myocytes, dyssynchronous Ca 2ϩ release was observed as the cells in culture dedifferentiated and lost TTs (18)...

The clustering of ryanodine receptors (RyR2) into functional Ca2+ release units is central to current models for cardiac excitation-contraction (E-C) coupling. Using immunolabeling and confocal microscopy, we have analyzed the distribution of RyR2 clusters in rat and ventricular atrial myocytes. The resolution of the three-dimensional structure was improved by a novel transverse sectioning method as well as digital deconvolution. In contrast to earlier reports, the mean RyR2 cluster transverse spacing was measured 1.05 microm in ventricular myocytes and estimated 0.97 microm in atrial myocytes. Intercalated RyR2 clusters were found interspersed between the Z-disks on the cell periphery but absent in the interior, forming double rows flanking the local Z-disks on the surface. The longitudinal spacing between the adjacent rows of RyR2 clusters on the Z-disks was measured to have a mean value of 1.87 microm in ventricular and 1.69 microm in atrial myocytes. The measured RyR2 cluster distribution is compatible with models of Ca2+ wave generation. The size of the typical RyR2 cluster was close to 250 nm, and this suggests that approximately 100 RyR2s might be present in a cluster. The importance of cluster size and three-dimensional spacing for current E-C coupling models is discussed.

Cardiac atrial cells lack a regular system of transverse tubules like that in cardiac ventricular cells. Nevertheless, many atrial cells do possess an irregular internal transverse‐axial tubular system (TATS). To investigate the possible role of the TATS in excitation‐contraction coupling in atrial myocytes, we visualized the TATS (labelled with the fluorescent indicator, Di‐8‐ANEPPS) simultaneously with Ca2+ transients and/or Ca2+ sparks (fluo‐4). In confocal transverse linescan images of field‐stimulated cells, whole‐cell Ca2+ transients had two morphologies: ‘U‐shaped’ transients and irregular or ‘W‐shaped’ transients with a varying number of points of origin of the Ca2+ transient. About half (54 %, n=289 cells, 13 animals) of the cells had a TATS. Cells with TATS had a larger mean diameter (13.2 ± 2.8 μm) than cells without TATS (11.7 ± 2.0 μm) and were more common in the left atrium (n= 206 cells; left atrium: 76 with TATS, 30 without TATS; right atrium: 42 with TATS, 58 without TATS). Simultaneous measurement of Ca2+ sparks and sarcolemmal structures showed that cells without TATS had U‐shaped transients that started at the cell periphery, and cells with TATS had W‐shaped transients that began simultaneously at the cell periphery and the TATS. Most (82 out of 102 from 31 cells) ‘spontaneous’ (non‐depolarized) Ca2+ sparks occurred within 1 μm of a sarcolemmal structure (cell periphery or TATS), and 33 % occurred within 1 pixel (0.125 μm). We conclude that the presence of a sarcolemmal membrane either at the cell periphery or in the TATS in close apposition to the sarcoplasmic reticulum is required for the initiation of an evoked Ca2+ transient and for spontaneous Ca2+ sparks.

To our knowledge, no data are available on whether the microbial species composition and abundance sampled with self-collected vaginal swabs are comparable to those of swabs collected by clinicians. Twenty healthy women were recruited to the study during a routine gynecological visit. Eligible women were between 18 and 40 years old with regular menstrual cycles. Participants self-collected a vaginal swab using a standardized protocol and then were examined by a physician, who collected an additional five swabs from the lateral wall of the mid-vagina. In this study, the self-collected and three physician-obtained swabs were analyzed and compared using terminal restriction fragment length polymorphism and sequence analyses of the 16S rRNA genes. Vaginal microbial community comparative statistical analyses of both T-RFLP and 16S rRNA gene sequence datasets revealed that self-collected vaginal swabs sampled the same microbial diversity as physician collected swabs of the mid-vagina. These findings enable large-scale, field-based studies of the vaginal microbiome.In recent years, "cultivation-independent" methods based on the analysis of 16S rRNA gene sequences directly extracted from biological samples are widely used to explore microbial diversity in various habitats (7,27,28). Using these methods, organisms are classified based on phylogenetic differences that are reflected in sequence polymorphisms of their 16S rRNA genes. The use of these approaches obviates the need to cultivate organisms, permits high-throughput analysis of samples, and provides precise and detailed information about the populations present. Using 16S rRNA gene analysis, the species (phylotype) composition and abundance in microbial communities can be readily determined, and similarities and differences among microbial communities can be quantitatively discerned. Despite difficulties in comparing data generated by different so-called "universal" PCR primer pairs, each introducing small biases (8, 36), this validated method has become the favored approach to characterizing the mutualistic microbial populations residing on and in the human body, including the gastrointestinal tract (4, 37), skin (4, 10), subgingival crevice (19), and vagina (4, 9, 15, 42). Importantly, the data obtained can be statistically analyzed to test the significance of changes that occur within individuals over time, or between individuals and treatment groups. These methods include terminal restriction fragment length polymorphisms (T-RFLP) (41) and high-throughput pyrosequencing of bar-coded 16S rRNA gene analysis (12,22).Surveys of vaginal microbial communities using cultivationindependent methods have been initiated under the NIH Roadmap Human Microbiome Project (14). Surveys of the vagina are important for a number of reasons. The beneficial effects of the endogenous microbiota on women's health in obstetric and gynecologic outcomes are numerous but poorly understood. A proper understanding of community membership, relative abundance, and variations therein are crit...