Evolution of exon-intron structure and alternative splicing in fruit flies and malarial mosquito genomes

Comparative analysis of alternative splicing of orthologous genes from fruit flies (Drosophila melanogaster and Drosophila pseudoobscura) and mosquito (Anopheles gambiae) demonstrated that both in the fruit fly genes and in fruit fly-mosquito comparisons, constitutive exons and splicing sites are more conserved than alternative ones. While >97% of constitutive D. melanogaster exons are conserved in D. pseudoobscura, only approximately 80% of alternative exons are conserved. Similarly, 77% of constitutive fruit fly exons are conserved in the mosquito genes, compared with <50% of alternative exons. Internal alternatives are more conserved than terminal ones. Retained introns are the least conserved, alternative acceptor sites are slightly more conserved than donor sites, and mutually exclusive exons are almost as conserved as constitutive exons. Cassette and mutually exclusive exons experience almost no intron insertions. We also observed cases of interconversion of various elementary alternatives, e.g., transformation of cassette exons into alternative sites. These results agree with the observations made earlier in human-mouse comparisons and demonstrate that the phenomenon of relatively low conservation of alternatively spliced regions may be universal, as it has been observed in different taxonomic groups (mammals and insects) and at various evolutionary distances.     

In vitro self-assembly of proepicardial cell aggregates: an embryonic vasculogenic model for vascular tissue engineering

Proepicardial/epicardial-derived cells are the main origin of the early embryonic coronary vascular bed. In vivo coronary vasculogenesis, which is a fast-occurring event, can be mimicked in vitro by culturing proepicardial tissue in different ways. The in vitro vasculogenic model presented in this study (a proepicardial suspension culture assay) partially reproduces coronary vascular development from its cellular precursors, a process known to be highly dependent on cell migration, cell differentiation, cell adhesion/sorting, and tissue fusion phenomena. The main aim of this study is to study the triggering signals and the cellular dynamics that regulate the differentiation of proepicardial cells into the angioblastic/endothelial lineage and their in vitro vasculogenic potential. Our results indicate that hanging drop-cultured proepicardia, which have an intrinsic vascular potential, behave like self-assembling cell aggregates or spheroids that can fuse to give rise to complex vascularized 3D structures. We believe that these self-assembling cell aggregates are an optimal choice to study the differentiation of coronary angioblasts, as well as a good method to reproduce vascular development in vitro. Finally, we propose the proepicardium as a suitable cellular source for vascular tissue engineering.     

A metabolic network in the evolutionary context: multiscale structure and modularity

The enormous complexity of biological networks has led to the suggestion that networks are built of modules that perform particular functions and are "reused" in evolution in a manner similar to reusable domains in protein structures or modules of electronic circuits. Analysis of known biological networks has revealed several modules, many of which have transparent biological functions. However, it remains to be shown that identified structural modules constitute evolutionary building blocks, independent and easily interchangeable units. An alternative possibility is that evolutionary modules do not match structural modules. To investigate the structure of evolutionary modules and their relationship to functional ones, we integrated a metabolic network with evolutionary associations between genes inferred from comparative genomics. The resulting metabolic-genomic network places metabolic pathways into evolutionary and genomic context, thereby revealing previously unknown components and modules. We analyzed the integrated metabolic-genomic network on three levels: macro-, meso-, and microscale. The macroscale level demonstrates strong associations between neighboring enzymes and between enzymes that are distant on the network but belong to the same linear pathway. At the mesoscale level, we identified evolutionary metabolic modules and compared them with traditional metabolic pathways. Although, in some cases, there is almost exact correspondence, some pathways are split into independent modules. On the microscale level, we observed high association of enzyme subunits and weak association of isoenzymes independently catalyzing the same reaction. This study shows that evolutionary modules, rather than pathways, may be thought of as regulatory and functional units in bacterial genomes.     

Nonmuscle myosin II powered transport of newly formed collagen fibrils at the plasma membrane

Collagen fibrils can exceed thousands of microns in length and are therefore the longest, largest, and most size-pleomorphic protein polymers in vertebrates; thus, knowing how cells transport collagen fibrils is essential for a more complete understanding of protein transport and its role in tissue morphogenesis. Here, we identified newly formed collagen fibrils being transported at the surface of embryonic tendon cells in vivo by using serial block face-scanning electron microscopy of the cell-matrix interface. Newly formed fibrils ranged in length from ∼1 to ∼30 µm. The shortest (1–10 µm) occurred in intracellular fibricarriers; the longest (∼30 µm) occurred in plasma membrane fibripositors. Fibrils and fibripositors were reduced in numbers when collagen secretion was blocked. ImmunoEM showed the absence of lysosomal-associated membrane protein 2 on fibricarriers and fibripositors and there was no effect of leupeptin on fibricarrier or fibripositor number and size, suggesting that fibricarriers and fibripositors are not part of a fibril degradation pathway. Blebbistatin decreased fibricarrier number and increased fibripositor length; thus, nonmuscle myosin II (NMII) powers the transport of these compartments. Inhibition of dynamin-dependent endocytosis with dynasore blocked fibricarrier formation and caused accumulation of fibrils in fibripositors. Data from fluid-phase HRP electron tomography showed that fibricarriers could originate at the plasma membrane. We propose that NMII-powered transport of newly formed collagen fibrils at the plasma membrane is fundamental to the development of collagen fibril-rich tissues. A NMII-dependent cell-force model is presented as the basis for the creation and dynamics of fibripositor structures.Cells have sophisticated mechanisms for transporting proteins from one location to another, often within membrane-bound vesicles that need to be of appropriate size and shape to accommodate the cargo. Collagen is a special case and is used as a model protein for studying protein transport; not only is collagen the most abundant structural protein in vertebrates, but it is too large to be accommodated within conventional transport vesicles. Moreover, collagen molecules self-assemble into structures of increasing size with each successive stage in the secretory pathway. The transported cargo increases from ∼0.5 MDa in the endoplasmic reticulum (ER) to several teradaltons (TDa) at the plasma membrane where the molecules are organized into fibrils. The motivation for our study was to build a temporal, spatial, and directional road map of the movement of membrane-bound collagen fibrils at the plasma membrane as the basis for a complete understanding of how cells transport collagen in the process of assembling a mechanically functional extracellular matrix (ECM).In brief, procollagen (the biosynthetic precursor of collagen) is synthesized in the ER and is asymmetric and relatively large; triple helical procollagen molecules are ∼1.5 nm diameter, ∼300 nm long, and have mass of ∼450 kDa. Tubular-saccular carriers containing procollagen arise from specialized ER domains (1, 2). TANGO1 recruits Sedlin and steers procollagen into COPII-dependent ER exit sites (3, 4) before CUL3-KLHL12 ubiquitinates a pool of sec31 molecules to drive procollagen transport from the ER to the Golgi apparatus (reviewed by ref. 5). ER-to-Golgi carriers join the Golgi stacks by fusing with cis cisternae and induce the formation of intercisternal tubules. Procollagen traverses the Golgi apparatus without leaving the cisternal lumen (6, 7). Once through the Golgi apparatus, procollagen can travel in pleomorphic Golgi-to-plasma membrane carriers (GPCs) that can be 0.3–1.7 µm in length (8). Evidence from electron microscopy suggests that these carriers contain sheaves of procollagen molecules stacked in zero register (9). Conversion of procollagen to collagen culminates in the formation of elongated fibrils that can be millimeters in length and are the primary tensile element of diverse tissues (10–12). Different types of collagen polymerize and form complexes with proteoglycans and other glycoproteins to form the largest assembles in vertebrate tissues (reviewed by ref. 13). The fibrils are deposited in embryonic tissues where they are ∼30 nm in diameter and contain ∼350 collagen molecules in the cross-section (based on calculations by ref. 14); thus, the fibrils are ∼0.3 TDa/µm. Considering that the first-formed fibrils are a few microns in length and grow to several millimeters, the fibrils are the largest size range structures transported by cells.The fact that collagen fibrils in vivo are too narrow and too densely packed to be resolved by light microscopy precludes real-time studies of cell–fibril interactions in vivo. Thus, transmission electron microscopy (TEM) has emerged as the method of choice for in vivo fibril studies but is not without its challenges because of the tortuosity and extreme lengths of the fibrils. Trelstad and Hayashi, and later Birk and Trelstad, used serial section transmission electron microscopy (ssTEM) to image the cell-matrix interface in embryonic tendon and were among the first to identify collagen fibrils in plasma membrane recesses (15, 16). Using data from an image stack of ∼14 µm depth (which is approximately one-fourth the length of an embryonic tendon cell; ref. 15), Trelstad and Hayashi proposed that the fibrils are formed by the addition of subassemblies of collagen onto the ends of the fibrils in plasma membrane recesses (15). In later studies, using image stacks of depth ∼20 µm, Canty and coworkers described actin-dependent slender projections (called fibripositors) that contained deeper recesses in which collagen fibrils were located (17, 18). The number of sections that can be cut for ssTEM limits the practicality of viewing entire cells and complete fibrils. However, serial block face-scanning electron microscopy (SBF-SEM; ref. 19) has emerged as a practical alternative to ssTEM for studying collagen fibril assembly in vivo (20).The experiments described below used SBF-SEM to study how cells transport collagen fibrils in vivo. We identified an abundance of short newly formed fibrils associated with a variety of plasma membrane morphologies and within intracellular fibricarriers; fibricarriers were identified that were larger than the GPCs and fibril vesicles that have been described previously. We show that collagen fibrils in fibricarriers and fibripositors decrease in abundance when procollagen secretion from the ER is blocked by dimethyloxaloylglycine (DMOG), which is a potent inhibitor of prolyl 4-hydroxylase (21). We show that fibricarriers can originate at the plasma membrane and are transported into fibricarriers powered by nonmuscle myosin II (NMII) in a process that is blocked by dynasore inhibition of dynamin-dependent endocytosis (22). We propose a unique transport pathway in which new collagen fibrils assembling on the cell surface are moved through the cell in a process powered by NMII.     

Lumbosacral pain syndrome in sportsmen and ballet-dancer (diagnostic algorithm)

There is presented an experience of the examination of 517 cases including sportsmen and ballet dancers, 15-45 olds, suffering from low back pain syndrome. 265 patients were diagnosed with osteochondrosis, 105 cases--with spondylolis, 92--with facet syndrome, and 55 cases--with pelvic ligaments ligamentopathia. Based upon the analysis of the results of examination diagnostic algorithm and differential diagnostics of these diseases were developed. The use of the proposed algorithm in patients with dissimilar variants of low back pain syndrome showed its informational content. It permits to make a right diagnosis and apply an adequate therapeutic approach.     

Posttraumatic posterior knee instability

At the department of the sport and ballet trauma new methods of arthroscopic management of posterior posttraumatic knee instability (posterior static stabilization of knee joint with using of single-banded and double-banded transplants) in dependence on the kind, degree and form of instability, were worked out and implemented into clinical practice. Such methods permit to attain a compensated or subcompensated form of knee joint stability. Ad hoc methods of management were used in 23 patients (20 males and 3 females). A total of 62 patients (51 males and 11 females) got operative treatment at the department, that permitted us to estimate fairly different methods of operative treatment of posttraumatic posterior knee joint instability. An operational intervention was performed in average 6 month after trauma. We received data in patients which had been treated with use of new methods of operative treatment as follows: good results were drawn in 93.3% (22 patients), satisfactory results--in 6.7% of cases (1 patient).     

Ultrastructural analysis of aortic endothelium after the host's death]

The changes of the endothelium of abdominal and thoracic aorta of rats and man (71 Wistar rats and 4 humans) during different periods after the death of the organism were analysed by means of scanning and transmission electron microscopy. Soon after death of the organism endotheliocytes are shortened and the cytoskeleton is structuralized. The changes result from hypoxia and the reaction develops within 60 minutes after death. Aortic endothelium is heteromorphic in its sensitivity to hypoxia. In the segments with high hemodynamic loads to the vessel wall the injury of endothelium develops more rapidly. The conversion from reversible to irreversible changes includes certain stages. In spite of different structure of rat and human aortic wall the stages of the endothelial injury after death and the time of beginning of irreversible injury are similar.     

Frequent alternative splicing of human genes

Alternative splicing can produce variant proteins and expression patterns as different as the products of different genes, yet the prevalence of alternative splicing has not been quantified. Here the spliced alignment algorithm was used to make a first inventory of exon-intron structures of known human genes using EST contigs from the TIGR Human Gene Index. The results on any one gene may be incomplete and will require verification, yet the overall trends are significant. Evidence of alternative splicing was shown in 35% of genes and the majority of splicing events occurred in 5' untranslated regions, suggesting wide occurrence of alternative regulation. Most of the alternative splices of coding regions generated additional protein domains rather than alternating domains.     

Opportunistic microorganisms in urinary tract diseases

The microflora of clinical material (urine, urethral discharge and smear, vaginal discharge and smear, feces in colonic dysbacteriosis) from patients with urinary tract infections (UTI) in the Moscow Region was studied. Escherichia coli and Staphylococcus epidermidis have been found to be the most common agents isolated from the urine of UTI patients. Pseudomonas aeruginosa and other staphylococcal species and enterococci account for a smaller proportion of infections. High- (E. coli and S. epidermidis) and moderate-priority pathogens are translocated into the urinary tract from the colonic biotope in dysbacteriosis. The causative agents of non-gonococcal urethritis are staphylococci (more commonly epidermal ones) in 50% of cases, those of pyelonephritis are staphylococci (predominantly E. coli) in more than 50% of cases. The etiological agents of female genital infections are staphylococci (mainly epidermal ones) in 40% of cases.     

Nocturnal and daytime panic attacks--comparison of sleep architecture, heart rate variability, and response to sodium lactate challenge.

BACKGROUND: The purpose of this study was to determine if nocturnal panic patients have greater autonomic dysregulation than patients with daytime panic. METHODS: Three groups were studied: patients who suffer from panic attacks during sleep (n = 12), those who suffer from daytime panic attacks only (n = 12), and control subjects (n = 12). Each subject underwent 24-hour holter monitoring for heart rate variability (HRV), an overnight sleep recording, and sodium lactate challenge during wakefulness. RESULTS: There was a marked subjective response to the sodium lactate challenge in the panic disorder (PD) patients but not in control subjects. Each group showed changes in HRV in response to sodium lactate challenge. The decrease in HRV measures was more marked in PD patients as a whole than in control subjects. During non-rapid eye movement (REM) sleep the value for total power (TP) was significantly higher in the nocturnal panic patients. The PD patients as a whole had higher values for TP and low-frequency (LF) power during REM sleep than control subjects. There were no significant differences between the two PD groups in sleep architecture. The PD patients as a whole had lower sleep efficiency and less stage 4 sleep than control subjects. CONCLUSIONS: These findings indicate that there are substantial differences between PD and control subjects in autonomic regulation and that there are small differences between patients with daytime panic attacks and those with sleep-related panic attacks.