Spatial recovery of the murine gut microbiota after antibiotics perturbation.

Bacterial communities are highly complex, with interaction networks dictating ecosystem function. Bacterial interactions are constrained by the spatial organization of these microbial communities, yet studying the spatial organization of microbial communities at the single-cell level has been technically challenging. Here, we use the recently developed high-phylogenetic-resolution microbiota mapping by fluorescence in situ hybridization technology to image the gut microbiota at the species and single-cell level. We simultaneously image 63 different bacterial species to spatially characterize the perturbation and recovery of the gut microbiota to ampicillin and vancomycin in the cecum and distal colon of mice. To decipher the biology in this complex imaging data, we developed an analytical framework to characterize the spatial changes of the gut microbiota to a perturbation. The three-tiered analytical approach includes image-level diversity, pairwise colocalization analysis, and hypothesis-driven neighborhood analysis. Through this workflow, we identify biogeographic and antibiotic-based differences in the spatial organization of the gut microbiota. We demonstrate that the cecal microbiota has increased micrometer-scale diversity than the colon at baseline and recovers better from perturbation. Also, we identify potential foundation and keystone species that have high baseline neighborhood richness and that are associated with recovery from antibiotics. Through this workflow, we add a spatial layer to the characterization of bacterial communities and progress toward a better understanding of bacterial interactions leading to improved microbiome modulation strategies. IMPORTANCE: Antibiotics have broad off-target effects on the gut microbiome. When the microbial community is unable to recover from antibiotics, it can lead to increased susceptibility to gastrointestinal infections and increased risk of immunological and metabolic diseases. In this study, we work to better understand how the gut microbiota recovers from antibiotics by employing a recent technology to image the entire bacterial community at once. Through this approach, we characterize the spatial changes in the gut microbiota after treatment with model antibiotics in both the cecum and colon of mice. We find antibiotic- and biogeographic-dependent spatial changes between bacterial species and that many of these spatial colocalizations do not recover to baseline levels even 35 days after antibiotic administration.

Serum integrin-linked kinase (sILK) concentration and survival in non-small cell lung cancer: a pilot study.

BACKGROUND: Integrin-linked kinase (ILK) is an intracellular signaling protein critically involved in cellular growth and motility. In non-small cell lung cancer (NSCLC), increased ILK expression has been associated with decreased recurrence-free and overall survival. Recently, ILK has also been detected in the serum of NSCLC patients. OBJECTIVE: To assess the prognostic impact of preoperative serum ILK (sILK) concentration on overall survival in surgically amenable NSCLC. PATIENTS AND METHODS: Preoperative sILK was quantified by ELISA in 50 newly diagnosed NSCLC patients. After surgery, patients were followed-up for a median interval of 2.5 years. RESULTS: Serum ILK concentrations ranged from 0 to 2.44 ng/ml. Mean sILK was around 2.3 times higher in the 16 patients who died as compared to the 34 patients who survived (1.04 vs. 0.45 ng/ml, p = 0.001). In univariate time-to-event analysis, increased sILK was associated with adverse survival [Hazard ratio (HR): 4.03, 95 % CI: 2.00-8.13, p < 0.001]. This association prevailed after multivariable adjustment for several clinical, demographic, and laboratory parameters (HR: 3.85, 95 % CI: 1.53-9.72, p = 0.004). CONCLUSIONS: Serum ILK shows potential as a novel strong and independent prognostic marker for postoperative survival in surgically amenable NSCLC.

Selective regulation of p38ß protein and signaling by integrin-linked kinase mediates bladder cancer cell migration.

Integrin-linked kinase (ILK) and p38(MAPK) are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38(MAPK) is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38(MAPK) is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38ß, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38ß or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38ß cellular protein level, without inhibiting p38ß messenger RNA (mRNA) expression. The loss of p38ß protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38ß. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK-p38ß complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38ß implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK-p38ß signaling complexes, playing a key role in bladder cancer cell migration.

Integrin-linked kinase: not so 'pseudo' after all.

Integrin-linked kinase (ILK) is a highly evolutionarily conserved intracellular protein that was originally identified as an integrin-interacting protein, and extensive genetic and biochemical studies have shown that ILK expression is vital during both embryonic development and tissue homeostasis. At the cellular and tissue levels, ILK regulates signaling pathways for cell adhesion-mediated cell survival (anoikis), apoptosis, proliferation and mitosis, migration, invasion, and vascularization and tumor angiogenesis. ILK also has central roles in cardiac and smooth-muscle contractility, and ILK dysregulation causes cardiomyopathies in humans. ILK protein levels are increased in several human cancers and often the expression level predicts poor patient outcome. Abundant evidence has accumulated suggesting that, of the diverse functions of ILK, some may require kinase activity whereas others depend on protein-protein interactions and are, therefore, independent of kinase activity. However, the past several years have seen an ongoing debate about whether ILK indeed functions as a protein serine/threonine kinase. This debate centers on the atypical protein kinase domain of ILK, which lacks some amino-acid residues thought to be essential for phosphotransferase activity. However, similar deficiencies are present in the catalytic domains of other kinases now known to possess protein kinase activity. Numerous studies have shown that ILK phosphorylates peptide substrates in vitro, corresponding to ILK-mediated phosphorylations in intact cells, and a recent report characterizing in vitro phosphotransferase activity of highly purified, full-length ILK, accompanied by detailed enzyme kinetic analyses, shows that, at least in vitro, ILK is a bona fide protein kinase. However, several genetic studies suggest that, not all biological functions of ILK require kinase activity, and that it can function as an adaptor/scaffold protein. Here, we review evidence for and against ILK being an active kinase, and provide a framework for strategies to further analyze the kinase and adaptor functions of ILK in different cellular contexts.

Integrin-linked kinase (ILK) is highly expressed in first trimester human chorionic villi and regulates migration of a human cytotrophoblast-derived cell line.

The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.

Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin-linked kinase, ILK1.

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1. Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments. Condi tional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM- and growth factor-stimulated ILK1 activity, but did not inhibit Raf-1 kinase activity. A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP-mediated inhibition of ILK1. Catalytically active, but not mutant ILKAP, strongly inhibited insulin-like growth factor-1-stimulated GSK3beta phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3beta signalling. Consistent with this, active, but not H154D mutant or the related PP2Calpha, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3beta phosphorylation.

Selection of viable cardiomyocytes for cell transplantation using three-dimensional tissue culture.

BACKGROUND: We developed a three-dimensional cardiac tissue culture system to select viable and contractile cells for the purpose of cardiomyocyte transplantation. In this study we will show that reaggregation of cardiomyocytes in culture is an active process indicative of cardiomyocyte viability and functionality. METHODS: Myocardial tissue from newborn mice has been enzymatically digested, incubated in culture inserts, and studied by phase contrast microscopy, conventional histology, immunohistochemistry, electron microscopy, and TUNEL assay. RESULTS: Cells that are plated on nonadhesive surfaces, reaggregate to spontaneously contracting cell aggregates. The capacity to reaggregate was significantly dependent from the age of the tissue donor (P<0.0001) and on the method of enzymatic dissociation (P<0.0001). The majority of cells within the aggregates consisted of cardiomyocytes. After 24 hr incubation, significant amounts of laminin and fibronectin had been deposited between the cells. Ultrastructural analysis revealed viable cardiomyocytes attached to each other by tight junctions. The apoptotic rate within the aggregates was 11.4+/-4.6 vs. 44.5+/-10.5% immediately after dissociation (P<0.05). CONCLUSIONS: The capacity to form spontaneously contracting aggregates is an inherent characteristic of viable cardiomyocytes in 3-dimensional cultures, which could be successfully exploited for cellular cardiomyocyte transplantation.

Auditory backward-masking performance by children who stutter and its relation to dysfluency rate.

The fluency of people who stutter is affected markedly when auditory feedback is altered, suggesting that stuttering may be associated with hearing. Peripheral hearing problems, however, are no more common in people who stutter than in those who do not. Performance was investigated in a task that involves central auditory processing (backward masking). Children who stuttered had deficits in backward masking (indicated by higher thresholds) compared with a group of fluent control children. The backward-masking thresholds were positively correlated with frequency of stuttering.

Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signalling.

Interaction of cells with the extracellular matrix (ECM) results in the regulation of cell growth, differentiation and migration by coordinated signal transduction through integrins and growth-factor receptors. Integrins achieve signalling by interacting with intracellular effectors that couple integrins and growth-factor receptors to downstream components. One well-studied effector is focal-adhesion kinase (FAK), but recently another protein kinase, integrin-linked kinase (ILK), has been identified as a receptor-proximal effector of integrin and growth-factor signalling. ILK appears to interact with and be influenced by a number of different signalling pathways, and this provides new routes for integrin-mediated signalling. This article discusses ILK structure and function and recent genetic and biochemical evidence about the role of ILK in signal transduction.

Mapping of the gene encoding the integrin-linked kinase, ILK, to human chromosome 11p15.5-p15.4.

We have recently reported the identification and cloning of the gene encoding p59ILK, a novel protein ser/thr kinase that is found in physiologic complexes with beta integrin subunits. ILK is a potential protoonocogene that appears to function in mediating signal transduction by beta 1 family integrins. Fluorescence in situ hybridization analysis of metaphase and decondensed free chromatin fibers localized ILK to 11p15.5-p15.4. This position was also confirmed by relational mapping using well-characterized translocations with breakpoints in chromosome band 11p15. Our results indicate that ILK maps between HBBC and CALC loci, in the 11p15.5-p15.4 band interval. This location may be important in evaluating the potential role of p59ILK in tumorigenesis since it has been shown that this region is associated with both genomic imprinting and loss of heterozygosity in certain types of tumor.

Rescue of a Drosophila NF1 mutant phenotype by protein kinase A.

The neurofibromatosis type 1 (NF1) tumor suppressor protein is thought to restrict cell proliferation by functioning as a Ras-specific guanosine triphosphatase-activating protein. However, Drosophila homozygous for null mutations of an NF1 homolog showed no obvious signs of perturbed Ras1-mediated signaling. Loss of NF1 resulted in a reduction in size of larvae, pupae, and adults. This size defect was not modified by manipulating Ras1 signaling but was restored by expression of activated adenosine 3', 5'-monophosphate-dependent protein kinase (PKA). Thus, NF1 and PKA appear to interact in a pathway that controls the overall growth of Drosophila.

Protein kinase mediators of integrin signal transduction.

Protein kinases are important mediators of signal transduction initiated by soluble growth factors and cytokines. Cellular interactions with the extracellular matrix are mediated largely by members of the integrin class of cell adhesion molecules, which also subsume signal transduction functions required for cell growth, differentiation, and survival. Here we review the involvement of protein kinases in mediating integrin intracellular signal transduction and the possible role for these molecules in regulating integrin adhesion. Although in most cases mechanistic details are incomplete, the emerging theme of protein kinases mediating cross-talk between growth factor receptor and integrin signalling systems provides a timely backdrop against which to present new developments in this area. The contribution of the actin cytoskeleton to integrin signal transduction is discussed, with respect to the concept of 'solid-state' signalling providing a mechanism for imposing order on the protein-protein interactions which underlie signal discrimination. Moreover, we review evidence that dysregulated integrin signalling contributes to pathological processes including arthritis, thrombasthenia, leucocyte adhesion deficiencies, and tumour angiogenesis and invasion.

Computers and medical information: an elective for fourth-year medical students.

The Computers and Medical Information elective is a collaborative effort to expose students to a variety of computer applications for medical information management. The course has a modular format so that students can work with a variety of people who are enthusiastic users of computer-based information systems. The elective emphasizes learning by doing. Faculty introduce concepts and systems and serve as guides in the use of systems. Students have rated the course positively and, after four years as an elective, some of the course's content has been integrated into the required curriculum.

Integrin cytoplasmic interactions and bidirectional transmembrane signalling.

Integrins are heterodimeric integral plasma membrane proteins containing extracellular, transmembrane, and cytoplasmic domains. These highly versatile receptors mediate not only cell adhesion and migration, but also the bidirectional transfer of information across the plasma membrane. The cytoplasmic domains of integrins are required for the transduction of this bidirectional information, and have recently been shown to participate in direct interactions with some novel cytoplasmic proteins, such as an ankyrin repeat containing serine/threonine protein kinase (integrin-linked kinase) and beta3 endonexin. New evidence also suggests that, via interactions with focal adhesion kinase, the integrin cytoplasmic domains can coordinate actin cytoskeletal organization and responses to growth factors. The elucidation of the signal transduction pathways activated by integrins is an intense area of investigation that has shown that integrins have some unique properties as signal transducing receptors.

Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase.

The interaction of cells with the extracellular matrix regulates cell shape, motility, growth, survival, differentiation and gene expression, through integrin-mediated signal transduction. We used a two-hybrid screen to isolate genes encoding proteins that interact with the beta 1-integrin cytoplasmic domain. The most frequently isolated complementary DNA encoded a new, 59K serine/threonine protein kinase, containing four ankyrin-like repeats. We report here that this integrin-linked kinase (ILK) phosphorylated a beta 1-integrin cytoplasmic domain peptide in vitro and coimmunoprecipitated with beta 1 in lysates of mammalian cells. Endogenous ILK kinase activity was reduced in response to fibronectin. Overexpression of p59ILK disrupted epithelial cell architecture and inhibited adhesion to integrin substrates, while inducing anchorage-independent growth. We propose that ILK is a receptor-proximal protein kinase regulating integrin-mediated signal transduction.

Diagnostic computing: an elective for fourth-year medical students.

The complex problems that patients pose require familiarity with the use and limitations of diagnostic computer systems. Independent of practice specialty, physicians may expect to encounter computer applications and digital image systems to provide information for diagnosis and management. This poster describes an elective for fourth-year medical students that introduces students to diagnostic computing through the use of decision-assist software and imaging processing systems. The poster includes photographs, examples of course exercises and readings, and a summary of course evaluation information.

Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases.

Mouse neurofibromatosis type 1 cDNA sequence reveals high degree of conservation of both coding and non-coding mRNA segments.

To identify evolutionary conserved domains and facilitate the recognition of potentially significant mutations in NF1 patients or tumors, we have determined the complete approximately 12 kb sequence of mouse neurofibromatosis type 1 mRNA. The sequence predicts a 2841 amino acid protein that is more than 98% identical to human neurofibromin. All but 9 of the 45 amino acid differences between mouse and human neurofibromin occur in the N-terminal half of the protein, with 16 changes clustered just upstream of the IRA-related segment. Given the high degree of sequence identity, virtually any sequence alteration in NF1 patients or tumors is potentially significant. We have also found that the 3' untranslated segment of NF1 mRNA is highly conserved, suggesting that this region may also be a target for mutations in NF1 patients.

Neurofibromatosis type 1 gene mutations in neuroblastoma.

The introduction of human chromosome 17 suppresses the tumourigenicity of a neuroblastoma cell line in the absence of any effects on in vitro growth and the neurofibromatosis type 1 (NF1) gene may be responsible. Here we report that 4 out of 10 human neuroblastoma lines express little or no neurofibromin and that two of these lines show evidence of NF1 mutations, providing further proof that NF1 mutations occur in tumours that are not commonly found in NF1 patients. We also show that NF1 deficient neuroblastomas show only moderately elevated ras-GTP levels, in contrast to NF1 tumour cells, indicating that neurofibromin contributes differently to the negative regulation of ras in different cell types.

Complete human NF1 cDNA sequence: two alternatively spliced mRNAs and absence of expression in a neuroblastoma line.

Neurofibromatosis type 1 (NF1) is caused by mutations in a large gene on chromosome 17q11.2. Previously described partial cDNAs for this gene predicted a protein related to yeast IRA1/IRA2 and the mammalian RAS GTPase activator protein GAP. To initiate a detailed study of the role of this gene in NF1, we have characterized a set of overlapping cDNAs that represent its complete coding sequence. Our results show that two differentially expressed human NF1 mRNAs differ by a 63-bp insertion in the GAP-related domain. These mRNAs predict two 2,818- and 2,839-amino acid proteins with calculated molecular masses of approximately 317 and 319 kD. Extensive similarity to IRA proteins is evident in a 1,450-amino-acid central segment, roughly between amino acids 900 and 2,350. However, the remainder of the NF1 protein is not significantly similar to other proteins. Interestingly, the SK-N-SH human neuroblastoma line expresses no detectable NF1 mRNA, indicating that expression of NF1 is not essential for viability of this neural crest-derived tumor cell line.