A Rare Case of Complete Inguinoscrotal Bladder Herniation With Ureteric Involvement: Assessing Diagnostic Challenges and Complex Surgical Management.

Inguinoscrotal hernias involving the urinary bladder are exceedingly rare, constituting a small subset of inguinal hernias. We present a case of a 47-year-old male with long-standing scrotal enlargement and obstructive uropathy due to complete herniation of the bladder with ureteric involvement. Diagnostic imaging confirmed the condition. Following an open laparotomy, the bladder was reduced, and a modified Bassini technique with orchiopexy was used for repair. Recurrence of the inguinoscrotal hernia with evidence of the bladder in the scrotal sac required additional surgery. This case underscores the rarity, diagnostic complexity, and potential complications of inguinoscrotal bladder hernias. Specialized surgical techniques and a multidisciplinary approach are crucial for successful management, especially in cases of complete bladder herniation. Future considerations should include innovative approaches to enhance primary repair outcomes for extensive hernias involving the bladder.

Cell cycle control by cell-matrix interactions.

Cell adhesion to the extracellular matrix (ECM) is required for normal cell cycle progression and accurate cell division. However, how cell adhesion to the wide range of ECM proteins found in human tissues influences the cell cycle is not fully understood. The composition and physical properties of the ECM can have profound effects on cell proliferation but can also promote cell cycle exit and quiescence. Furthermore, during tumor development and progression, changes in the ECM can drive both cancer cell proliferation and dormancy. Cell-matrix adhesion is primarily sensed via integrin-associated adhesion complexes, which in turn are regulated by the cell cycle machinery. In particular, cyclin-dependent kinase 1 (CDK1) has been shown to play a crucial role in regulating adhesion complexes during interphase and entry into mitosis. These reciprocal links between cell cycle progression and cell-matrix interactions are now being identified.

Giant worm-shaped ESCRT scaffolds surround actin-independent integrin clusters.

Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.

Pancreatic ductal adenocarcinoma cells employ integrin α6ß4 to form hemidesmosomes and regulate cell proliferation.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6ß4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin ß4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6ß4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1.

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms.

Connections between the cell cycle, cell adhesion and the cytoskeleton.

Cell division, the purpose of which is to enable cell replication, and in particular to distribute complete, accurate copies of genetic material to daughter cells, is essential for the propagation of life. At a morphological level, division not only necessitates duplication of cellular structures, but it also relies on polar segregation of this material followed by physical scission of the parent cell. For these fundamental changes in cell shape and positioning to be achieved, mechanisms are required to link the cell cycle to the modulation of cytoarchitecture. Outside of mitosis, the three main cytoskeletal networks not only endow cells with a physical cytoplasmic skeleton, but they also provide a mechanism for spatio-temporal sensing via integrin-associated adhesion complexes and site-directed delivery of cargoes. During mitosis, some interphase functions are retained, but the architecture of the cytoskeleton changes dramatically, and there is a need to generate a mitotic spindle for chromosome segregation. An economical solution is to re-use existing cytoskeletal molecules: transcellular actin stress fibres remodel to create a rigid cortex and a cytokinetic furrow, while unipolar radial microtubules become the primary components of the bipolar spindle. This remodelling implies the existence of specific mechanisms that link the cell-cycle machinery to the control of adhesion and the cytoskeleton. In this article, we review the intimate three-way connection between microenvironmental sensing, adhesion signalling and cell proliferation, particularly in the contexts of normal growth control and aberrant tumour progression. As the morphological changes that occur during mitosis are ancient, the mechanisms linking the cell cycle to the cytoskeleton/adhesion signalling network are likely to be primordial in nature and we discuss recent advances that have elucidated elements of this link. A particular focus is the connection between CDK1 and cell adhesion. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

Reticular adhesions are a distinct class of cell-matrix adhesions that mediate attachment during mitosis.

Adhesion to the extracellular matrix persists during mitosis in most cell types. However, while classical adhesion complexes, such as focal adhesions, do and must disassemble to enable mitotic rounding, the mechanisms of residual mitotic cell-extracellular matrix adhesion remain undefined. Here, we identify 'reticular adhesions', a class of adhesion complex that is mediated by integrin αvß5, formed during interphase, and preserved at cell-extracellular matrix attachment sites throughout cell division. Consistent with this role, integrin ß5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations. Reticular adhesions are morphologically and dynamically distinct from classical focal adhesions. Mass spectrometry defines their unique composition, enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding proteins but lacking virtually all consensus adhesome components. Indeed, reticular adhesions are promoted by PtdIns(4,5)P2, and form independently of talin and F-actin. The distinct characteristics of reticular adhesions provide a solution to the problem of maintaining cell-extracellular matrix attachment during mitotic rounding and division.

Cell adhesion is regulated by CDK1 during the cell cycle.

In most tissues, anchorage-dependent growth and cell cycle progression are dependent on cells engaging extracellular matrices (ECMs) via integrin-receptor adhesion complexes. In a highly conserved manner, cells disassemble adhesion complexes, round up, and retract from their surroundings before division, suggestive of a primordial link between the cell cycle machinery and the regulation of cell adhesion to the ECM. In this study, we demonstrate that cyclin-dependent kinase 1 (CDK1) mediates this link. CDK1, in complex with cyclin A2, promotes adhesion complex and actin cytoskeleton organization during interphase and mediates a large increase in adhesion complex area as cells transition from G1 into S. Adhesion complex area decreases in G2, and disassembly occurs several hours before mitosis. This loss requires elevated cyclin B1 levels and is caused by inhibitory phosphorylation of CDK1-cyclin complexes. The inactivation of CDK1 is therefore the trigger that initiates remodeling of adhesion complexes and the actin cytoskeleton in preparation for rapid entry into mitosis.

A Novel Technique to Repair Acute and Chronic Proximal Hamstring Avulsions.

Treatment of proximal hamstring avulsions is challenging regarding both timing and choice of repair. In chronic situations, the tissue can become attenuated, bringing into question the need for adjunctive allograft. In general, proximal hamstring avulsions are affixed back to the ischial tuberosity with suture anchors, with allograft used as needed for chronic situations. This article reports on a novel technique of suspensory fixation with a unicortical button but without allograft to repair both acute and chronic proximal hamstring avulsions. Three patients, all of whom regained full strength and functionality 1 year postoperatively, are described. [Orthopedics. 2018; 41(2):116-119.].

Defining the phospho-adhesome through the phosphoproteomic analysis of integrin signalling.

Cell-extracellular matrix (ECM) adhesion is a fundamental requirement for multicellular existence due to roles in positioning, proliferation and differentiation. Phosphorylation plays a major role in adhesion signalling; however, a full understanding of the phosphorylation events that occur at sites of adhesion is lacking. Here we report a proteomic and phosphoproteomic analysis of adhesion complexes isolated from cells spread on fibronectin. We identify 1,174 proteins, 499 of which are phosphorylated (1,109 phosphorylation sites), including both well-characterized and novel adhesion-regulated phosphorylation events. Immunoblotting suggests that two classes of phosphorylated residues are found at adhesion sites-those induced by adhesion and those constitutively phosphorylated but recruited in response to adhesion. Kinase prediction analysis identifies novel kinases with putative roles in adhesion signalling including CDK1, inhibition of which reduces adhesion complex formation. This phospho-adhesome data set constitutes a valuable resource to improve our understanding of the signalling mechanisms through which cell-ECM interactions control cell behaviour.

Paxillin kinase linker (PKL) regulates Vav2 signaling during cell spreading and migration.

The Rho family of GTPases plays an important role in coordinating dynamic changes in the cell migration machinery after integrin engagement with the extracellular matrix. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase-activating proteins (GAPs). However, the mechanisms by which GEFs and GAPs are spatially and temporally regulated are poorly understood. Here the activity of the proto-oncogene Vav2, a GEF for Rac1, RhoA, and Cdc42, is shown to be regulated by a phosphorylation-dependent interaction with the ArfGAP PKL (GIT2). PKL is required for Vav2 activation downstream of integrin engagement and epidermal growth factor (EGF) stimulation. In turn, Vav2 regulates the subsequent redistribution of PKL and the Rac1 GEF ß-PIX to focal adhesions after EGF stimulation, suggesting a feedforward signaling loop that coordinates PKL-dependent Vav2 activation and PKL localization. Of interest, Vav2 is required for the efficient localization of PKL and ß-PIX to the leading edge of migrating cells, and knockdown of Vav2 results in a decrease in directional persistence and polarization in migrating cells, suggesting a coordination between PKL/Vav2 signaling and PKL/ß-PIX signaling during cell migration.

Beta2-adaptin binds actopaxin and regulates cell spreading, migration and matrix degradation.

Cell adhesion to the extracellular matrix is a key event in cell migration and invasion and endocytic trafficking of adhesion receptors and signaling proteins plays a major role in regulating these processes. Beta2-adaptin is a subunit of the AP-2 complex and is involved in clathrin-mediated endocytosis. Herein, ß2-adaptin is shown to bind to the focal adhesion protein actopaxin and localize to focal adhesions during cells spreading in an actopaxin dependent manner. Furthermore, ß2-adaptin is enriched in adhesions at the leading edge of migrating cells and depletion of ß2-adaptin by RNAi increases cell spreading and inhibits directional cell migration via a loss of cellular polarity. Knockdown of ß2-adaptin in both U2OS osteosarcoma cells and MCF10A normal breast epithelial cells promotes the formation of matrix degrading invadopodia, adhesion structures linked to invasive migration in cancer cells. These data therefore suggest that actopaxin-dependent recruitment of the AP-2 complex, via an interaction with ß2-adaptin, to focal adhesions mediates cell polarity and migration and that ß2-adaptin may control the balance between the formation of normal cell adhesions and invasive adhesion structures.

Different roles of G protein subunits beta1 and beta2 in neutrophil function revealed by gene expression silencing in primary mouse neutrophils.

Neutrophils play important roles in host innate immunity and various inflammation-related diseases. In addition, neutrophils represent an excellent system for studying directional cell migration. However, neutrophils are terminally differentiated cells that are short lived and refractory to transfection; thus, they are not amenable for existing gene silencing techniques. Here we describe the development of a method to silence gene expression efficiently in primary mouse neutrophils. A mouse stem cell virus-based retroviral vector was modified to express short hairpin RNAs and fluorescent marker protein at high levels in hematopoietic cells and used to infect mouse bone marrow cells prior to reconstitution of the hematopoietic system in lethally irradiated mice. This method was used successfully to silence the expression of Gbeta(1) and/or Gbeta(2) in mouse neutrophils. Knockdown of Gbeta(2) appeared to affect primarily the directionality of neutrophil chemotaxis rather than motility, whereas knockdown of Gbeta(1) had no significant effect. However, knockdown of both Gbeta(1) and Gbeta(2) led to significant reduction in motility and responsiveness. In addition, knockdown of Gbeta(1) but not Gbeta(2) inhibited the ability of neutrophils to kill ingested bacteria, and only double knockdown resulted in significant reduction in bacterial phagocytosis. Therefore, we have developed a short hairpin RNA-based method to effectively silence gene expression in mouse neutrophils for the first time, which allowed us to uncover divergent roles of Gbeta(1) and Gbeta(2) in the regulation of neutrophil functions.

VEGFR1 (Flt1) regulates Rab4 recycling to control fibronectin polymerization and endothelial vessel branching.

The cell's main receptor for VEGF, VEGFR2 (Kdr) is one of the most important positive regulators of new blood vessel growth and its downstream signalling is well characterized. By contrast, VEGFR1 (Flt1) and the mechanisms by which this VEGF receptor promotes branching morphogenesis in angiogenesis remain relatively unclear.Here we report that engagement of VEGFR1 activates a Rab4A-dependent pathway that transports alphavbeta3 Integrin from early endosomes to the plasma membrane, and that this is required for VEGF-driven fibronectin polymerization in endothelial cells. Furthermore, VEGFR1 acts to promote endothelial tubule branching in an organotypic model of angiogenesis via a mechanism that requires Rab4A and alphavbeta3 Integrin. We conclude that a recycling pathway regulated by Rab4A is a critical effector of VEGFR1 during branching morphogenesis of the vasculature.

The DNA sequence of the human X chromosome.

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.