Climate and cyclic hydrobiological changes of the Barents Sea from the twentieth to twenty-first centuries

The Barents Sea is a transition zone between North Atlantic and Arctic waters, so its marine ecosystem is highly sensitive to climate dynamics. Understanding of marine biota response to climate changes is necessary to assess the environmental stability and the state of marketable biological resources. These processes are analyzed using a database from the Murmansk Marine Biological Institute which holds oceanographic and hydrobiological data sets collected for more than 100?years along the meridional Kola Transect in the Barents Sea. The data demonstrate high variability in thermal state of the upper layer of the Barents Sea, which is regulated by varying the inflow of Atlantic water and by regional climate. At irregular intervals, cold periods with extended seasonal ice cover are followed by warm periods. The most recent warm period started in the late 1980s and reached its maximum from 2001 to 2006. These cyclic changes in hydrologic regime across the twentieth century and first decade of the twenty-first century are reflected (with a specific lag of 1–5?years) by changes in species composition, as well as abundance and distribution of boreal and arctic groups of macrozoobenthos and fish fauna. For instance, cod and cod fisheries in the Barents Sea are closely linked to the marine climate. Furthermore, Kamchatka crab stock recruitment benefited from the warm climate of 1989 and 1990. In general, studies in this region have shown that climatic dynamics may be assessed using biological indices of abundance, biomass, and migration of marine organisms, including commercial species.     

Effect of salinity change on cardiac activity in Hiatella arctica and Modiolus modiolus, in the White Sea

A great majority of salinity studies have dealt with intertidal species. Little is known about the way subtidal animals respond to salinity fluctuations. Even less details are available on invertebrates from the White Sea, which salinity is ca. 25. The heart rate of two subtidal Bivalvia—Hiatella arctica and Modiolus modiolus—exposed to different salinities was recorded. Changes in cardiac activity were monitored for 9 days of the animals’ acclimation to salinities of 15, 20, 30 and 35, and for 4 days of reacclimation (return to the initial salinity of 25). The initial response to salinity change was a significant heart rate reduction. On the other hand, cardiac activity in M. modiolus intensified at salinities of 30 and 35. Reacclimation induced different HR responses: from a decrease to a rise, depending on the species and the salinity applied in the experiment. The differences in responses to salinity are discussed with respect to the morphological and ecological characteristics of the species.     

Conformational flexibility of RecA protein filament: transitions between compressed and stretched states

RecA protein is a central enzyme in homologous DNA recombination, repair and other forms of DNA metabolism in bacteria. It functions as a flexible helix-shaped filament bound on stretched single-stranded or double-stranded DNA in the presence of ATP. In this work, we present an atomic level model for conformational transitions of the RecA filament. The model describes small movements of the RecA N-terminal domain due to coordinated rotation of main chain dihedral angles of two amino acid residues (Psi/Lys23 and Phi/Gly24), while maintaining unchanged the RecA intersubunit interface. The model is able to reproduce a wide range of observed helix pitches in transitions between compressed and stretched conformations of the RecA filament. Predictions of the model are in agreement with Small Angle Neutron Scattering (SANS) measurements of the filament helix pitch in RecA::ADP-AlF(4) complex at various salt concentrations.     

Potent 4-amino-5-azaindole factor VIIa inhibitors

The 4-amino-5-azaindole as an amidino-benzimidazole replacement is described. A series of potent and selective analogs were discovered and showed desirable ex vivo efficacy as measured by PT.     

Generation of potent coagulation protease inhibitors utilizing zinc-mediated chelation

Inhibition of coagulation proteases such as thrombin, fXa, and fVIIa has been a focus of ongoing research to produce safe and effective antithrombotic agents. Herein, we describe a unique zinc-mediated chelation strategy to streamline the discovery of potent inhibitors of fIIa, fXa, and fVIIa. SAR studies that led to the development of selective inhibitors of fXa will also be detailed.     

Factor VIIa inhibitors: gaining selectivity within the trypsin family

Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challenging. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1) scaffold with potency for fVIIa and high selectivity against factors IIa, Xa, and trypsin. With this scaffold class, we propose that a unique hydrogen bond interaction between a hydroxyl on the distal ring of the biaryl system and the backbone carbonyl of fVIIa lysine-192 provides a basis for enhanced selectivity and potency for fVIIa.     

Aminopiperidine-fused imidazoles as dipeptidyl peptidase-IV inhibitors

A new series of DPP-4 inhibitors derived from piperidine-fused benzimidazoles and imidazopyridines is described. Optimization of this class of DPP-4 inhibitors led to the discovery of imidazopyridine 34. The potency, selectivity, cross-species DMPK profiles, and in vivo efficacy of 34 is reported.     

Collective Cell Migration Drives Morphogenesis of the Kidney Nephron

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis.     

Fluid flow and guidance of collective cell migration

Collective cell migration is emerging as a significant component of many biological processes including metazoan development, tissue maintenance and repair and tumor progression. Different contexts dictate different mechanisms by which migration is guided and maintained. In vascular endothelia subjected to significant shear stress, fluid flow is utilized to properly orient a migrating group of cells. Recently, we discovered that the developing zebrafish pronephric epithelium undergoes a similar response to luminal fluid flow, which guides pronephric epithelial migration towards the glomerulus. Intratubular migration leads to significant changes in kidney morphology. This novel process provides a powerful in vivo model for further exploration of the mechanisms underlying mechanotransduction and collective migration.Key words: collective migration, fluid flow, mechanotransduction, development, kidney, zebrafishThe term “collective cell migration” (collective motion) was first introduced to describe the behavior of starved Dictyostelium discoideum.¹ The term has rapidly gained general acceptance as encompassing a wide variety of coordinated cell migratory behaviors. A number of definitions have been proposed to unify the various collective migratory behaviors. Friedl et al.² defined it as “the movement of cell groups, sheets or strands consisting of multiple cells that are mobile yet simultaneously connected by cell-cell junctions.” This definition implies a number of features setting collective migration apart from other migratory behaviors. First, it points to the spatial restrictions on the individual cells within the migrating groups. The cells cannot leave the group and continue on their own. Therefore, they must respect the behavior of their neighbors and the overall migration occurs through the integration of individual cell activities across the collective. Second, it implies that different cells within the migrating group may play different roles. Some of them may not be migratory at all and simply “ride” the rest of the group, as indeed seen in border cell migration.³ Other cells within the group may further specialize into leaders and followers as can be seen in most current models of collective migration.⁴A variety of biological processes satisfy this definition. They include, among others, closure of wounded epithelial sheaths,³ physiological maintenance of intestinal epithelium,⁵ cancer invasion,^2,4,6 developmental processes of branching morphogenesis,^7,8 vascular sprouting,⁹ gastrulation,¹⁰ dorsal embryo closure,¹¹ as well as movements of some basal metazoan organisms such as sponges.¹² Over the years, a number of models emerged to study the process of collective migration.When starved, thousands of single cells of Dictyostelium discoideum aggregate and form a “slug” that migrates to the soil surface to form a fruiting body. This process has two general stages: the stage of aggregation, where individual migrating cells respond to cAMP concentration to form a multicellular aggregate¹³ and the stage of collective migration. In the latter stage, the leading (pre-stalk) cells of the slug secrete cAMP. In addition, they produce slime sheath that provides traction support for the aggregate. The slime sheath allows outermost cells of the aggregate to develop necessary traction for the entire slug to propel itself towards guidance cues. A number of molecular and cellular components have been recently identified to be important in this process, including integrin-, paxillin-like molecules and dynamic focal adhesion formation.¹⁴ Thus, Dictyostelium serves as a useful model for understanding the dynamic mechanisms of force formation in a migrating collective.Another well-established model of collective cell migration is the migration of border cells during ovary development in Drosophila. There, a small group of cells consisting of a central pair of polar cells surrounded by migratory outer border cells delaminate from the epithelium and migrate as a free group between nurse cells. Because of the tight nature of the migrating group, non-motile polar cells as well as mutant outer migratory cells can be carried within the group by their migratory companions.³ The migrating cluster uses nurse cells to generate the necessary traction to continue along the migratory path and rely on E-cadherin to accomplish this task.¹⁵ It has been proposed that the migrating border cell cluster is guided collectively wherein each outer border cell is inherently polarized, having an outer aspect and the inner aspect, so that the net migration of the cluster is simply the net of all the forces generated by the outer collective.¹⁶ It has been shown recently that both individual cell guidance and the collective cell guidance are at play in border cell migration.¹⁷Perhaps the best-studied examples of collective cell migration are found in the wound closure of epithelial sheets. Both kidney and gastric epithelial cell lines have been extensively studied in the wound closure assay to reveal important details of the collective migration that is a central process in wound repair. Recent studies have demonstrated the role of integrins, Rac, ERK, MAPK, Src and Pi3K among others as important molecular components of this processes.^18–21 A recent siRNA screen using breast epithelial cells identified a number of molecules that either inhibit or augment epithelial migration.²² This study revealed 42 genes previously unknown to be involved in migration. Many genes clustered within β-catenin, β1-integrin and actin networks in secondary analysis.While in vitro epithelial wound assays continue to provide insights into potential mechanisms of collective cell migration, the most developed in vivo vertebrate model comes from the studies of the zebrafish lateral line formation. In this process, the lateral line primordium cells move as a group in the anterior-posterior direction.^4,23 The migration is dependent on the interaction of stromal factor Cxcl12 along the guidance path and its receptor Cxcr4b.²⁴ The direction of migration is defined by the interplay between Fgf and Wnt signaling (rear and front of the migrating group, respectively). Wnt signaling in the front of the migrating lateral line inhibits Cxcr7b expression and promotes Cxcr4b expression. It also results in the secretion of Fgf ligands. Expression of sef at the front (also under control of Wnt) prevents Fgf from acting in this front domain. Fgf ligants interact with their receptors in the trailing end of the migrating group. As a result, the cells at the trailing end express dkk1 (to limit Wnt signaling) and Cxcr7b while downregulating Cxcr4b.^4,23,25,26 Thus, Cxcl12-Cxcr4b interaction is limited to the migration front. Cxcr7b expressed in the back of the migrating collective is believed to further interfere with Cxcl12-Cxcr4b interaction by sequestering Cxcl12. The net result of the differential signaling is the establishment of a distinct migratory front at the posterior aspect of the precursor population. At the same time, groups of cells at the back stop migrating and give rise to individual lateral line organs.The existence of a distinct migratory front is a unifying feature of all the models of collective migration described above. The migratory front defines the interface between the migratory collective and the tissues into which the migratory group advances. The front may be maintained by a stable pattern of signaling within the migrating group, as seen in lateral line migration where Wnt signaling at the front and FGF signaling at the back are maintained through mutual exclusion. Alternatively, the front may be maintained through spatial differences in concentrations of chemoattractants rendering the front of the group more migratory, as seen in the Drosophila border cell migration.³ In other systems, the migratory front may be maintained through cell-to-cell direct signaling, such as Notch signaling in determining the tips of vascular sprouts.⁹ Furthermore, migrating epithelial cultures in wound assays are inherently polarized by the presence of a free margin. Interestingly, the presence of the margin, which becomes the migratory front, is sufficient even in the absence of the wound to initiate a directed migration.²⁷ However, several new studies revealed that the existence of a distinct migratory front is not a universal or required feature of collective migration.^28–31Recently we discovered a novel form of collective migration that guides the morphogenesis and maturation of pronephric kidney.²⁸ The zebrafish pronephros is a simple bilaterally symmetrical structure consisting of two fused glomeruli, each connected to a pronephric tubule that runs posteriorly, eventually exiting at the level of the cloaca. The pronephros begins to function shortly after 1 dpf.²⁸ After the onset of its function, a signifi- cant maturation of the pronephros takes place, manifested at the structural level by the development of proximal convolution and re-positioning of nephron segment boundaries (Fig. 1). We demonstrated that both of these structural changes are a direct consequence of the collective epithelial migration that starts at about 30 hpf and lasts for the next three days. This proximal migration is governed by the onset of luminal fluid flow. The cells of the pronephric epithelium move enmasse towards the glomerulus and against the flow of urine. As a result, the proximal segment becomes compressed, shortened and convoluted. In contrast, the distal segment straightens and becomes longer (Fig. 1 and Suppl. Movie 1). This lengthening of the distal kidney is accompanied by cell proliferation that compensates for the proximal shift of kidney segments and allows for the en-masse migration to continue for three days.²⁸Open in a separate windowFigure 1Effect of pronephric migration on tubule architecture. (A) Schematic representation of zebrafish showing the pronephric kidney. Arrowhead points to the glomerulus. Arrow points to the pronephric tubule. (B) Pronephric architecture at 1 dpf before the onset of tubule flow. Dark shading indicates a distal segment. (C) Pronephric architecture at 3 dpf showing a markedly shortened and folded (convoluted) proximal segment. (D) Pronephric architecture at 3 dpf in the embryo with eliminated glomerular filtration. The position of the tubule convolution is now at the interface of the proximal and the distal tubules (see also Suppl. movie files 1 and 2).As mentioned above, this novel developmental process differs from most models of collective cell migration in at least one aspect; it lacks a distinct migratory front. In the absence of such front, the polarity of the migrating pronephric epithelium is established by using fluid flow as the guiding cue. When directed fluid flow is eliminated by obstructing the pronephros, the proximal migration is disrupted. Instead, the cells of the pronephric epithelium can often be seen migrating circumferentially, around the tube perimeter. This circumferential pseudo-migration correlates with the presence of local vortex currents in obstructed pronephroi due to the presence of beating cilia. Indeed, we failed to observe similar circumferential pseudomigratory behavior in paralyzed cilia mutants (unpublished data). In addition, we were able to engineer an ectopic convolution (about 500 µm distal to its normal location next to the glomerulus) by selectively eliminating proximal, but not distal sources of fluid flow (Fig. 1 and Suppl. Movie 2). This finding further supports the conclusion that luminal fluid flow guides the epithelial migration. It is still possible that different cells within the pronephric epithelium have distinct roles in orchestrating the migration. For instance, a small subset of cells could act as functional leaders and organize the migration process. Alternatively, luminal flow could directly interact with each migrating cell. Further studies should determine which scenario is present in the pronephros.There are at least two other systems where cell migration is governed by the mechanical forces generated by luminal fluid flow. Vascular endothelial cells respond to fluid shear stress, orient in the direction of the flow and migrate in the direction of shear force. This behavior is thought to be important in vascular remodeling.²⁹ A related model was developed in macaque placental trophoblast cells which demonstrate a similar behavior.³⁰ It is notable that in a wound assay, endothelial cells respond in a way similar to that in other in vitro wound models described above.³² Thus, more than one mode of guidance may be present in a given tissue.Significant advances have been made in our understanding of the cellular responses to shear stress in vascular endothelium. Endothelial cells sense and respond to fluid shear by utilizing a system of adhesion molecules including PECAM and VE-cadherin, integrin activation, activation of VEGFR, calcium influx, and modulation of the cytoskeleton by Rho family GTPases.^29,31 Recent evidence also suggests that sensory cilia play a role in the endothelial response to shear stress.^33,34 Fluid shear first induces lamellipodial cell extensions, followed by basal protrusions and new focal adhesion formation in the direction of the flow. Subsequent migration requires remodeling of adhesions and release of cell substratum attachments at the rear of the migrating cell.Migration of pronephric epithelial cells is likely to involve similar basic mechanisms. For instance, we have observed a strong correlation between the presence of directed lamellipodial extensions of epithelial cells on the tubule basement membranes and the basal phosphoFAK staining, suggesting that pronephric epithelial cells actively remodel their matrix attachments as they migrate. The similarities and differences between these two systems are likely to prove useful in determining how mechanical forces establish self-perpetuating cell movement. A notable difference between the pronephric cell migration and endothelial cell migration is that pronephric cells migrate against the flow as opposed to in the direction of the flow, suggesting that the exact nature of the process linking flow to migration may also be different.Several mechanisms may be at play in transducing the directional flow into directed migration of pronephric epithelia. First, ciliary function has been implicated in sensing fluid flow.³⁵ Thus, it is possible that bending the luminal cilia is key to the translation of luminal flow into collective epithelial cell migration. However, this potential mechanism was not supported by our observations. In particular, we tested the role of polycystins that are thought to be central to the flow sensing mechanism in the cilia.^33,36 We observed that polycystin morphants did not show an arrest or misorientation of migration until pronephric cycts were formed. This finding indicates that polycystins affect migration secondarily, due to pronephric obstruction and perturbation of flow, rather than by directly influencing epithelial migration. While polycystins do not appear to mediate mechanotransduction in pronephric epithelial migration, other members of the TRP ion channel family may be involved. Many TRP channels, such as TRPV, TRPC as well as other mechanosensitive ion channels, are thought to mediate transduction of mechanical stimuli into the intracellular signals.^37,38 It is possible that one or more such mechanisms are present in the pronephros.Alternatively, as discussed above, shear stress may be transduced at focal adhesions through integrin coupled intracellular signals with multiple potential intracellular targets, including Src, FAK, ILK, paxillin and p130Cas.³⁹ It has been shown, for example, that in cultured intestinal epithelial cells, a mechanical deformation of the substrate stimulates migration in FAK dependent manner.⁴⁰ Cell-cell junctions may also serve as a major site of mechanotransduction as was shown in vascular endothelia.³¹Other potential components of mechanotransduction include G-protein coupled receptors, which were shown to localize to the sites of focal adhesion and are known to be activated by shear stress and cyclic stretching.³⁸ Here, mechanical displacement may lead to the conformational change in the receptor molecule and the activation of downstream targets. In addition, Wnt and receptor tyrosine kinase signaling have been linked to mechanotransduction.^38,41 It remains to be determined which of these processes mediate the relation between pronephric flow and epithelial migration.It is possible, however, that multiple components (focal adhesion complexes, cell junctions, sensory cilia, etc.) interact with each other, and these interactions are integrated by the cell to generate a response to a mechanical stimulus. There is evidence showing that various components are indeed linked together by cytoskeleton.^34,42 The apical ciliary response to shear stress by cultured kidney cells (as measured by the cytoplasmic calcium increase) can be prevented by altering the integrity and the tensile properties of the cytoskeleton. The same result can be achieved by blocking the integrin interaction with extracellular matrix at the basal surface.⁴² Conversely, disrupting ciliary function in vascular endothelial cells significantly attenuates the overall response of the cell to fluid shear, the result that can also be achieved by disrupting cytoplasmic microtubule polymerization.³⁴These findings suggest a model in which multiple identified components of the mechanotransduction response are linked by cytoskeletal elements, that allow events at each specific location to influence the state of a different remote cell component directly.⁴³ For example, bending the cilium would have opposite mechanical effects on cell-cell junctions located in the direction of the bending, compared to those located on the opposite side.³⁴ Importantly, this mechanical communication is inherently bidirectional and would allow the cell to instantly integrate signals originating at different locations and initiate a robust and coordinated response to external mechanical cues.