Saliva-controlled sputum culture (SSC) - A high value diagnostic tool for deep pulmonary infections.

A major obstacle to prompt diagnosis of fungal pulmonary infections is that deep sputum samples are scarce, yet are frequently rejected if they contain saliva. We show that including saliva controls unfailingly distinguishes oropharyngeal flora from pulmonary fungi, thus preserving valuable samples for analysis, expediting diagnoses and improving patient care.

A Twist-Snail axis critical for TrkB-induced epithelial-mesenchymal transition-like transformation, anoikis resistance, and metastasis.

In a genomewide anoikis suppression screen for metastasis genes, we previously identified the neurotrophic receptor tyrosine kinase TrkB. In mouse xenografts, activated TrkB caused highly invasive and metastatic tumors. Here, we describe that TrkB also induces a strong morphological transformation, resembling epithelial-mesenchymal transition (EMT). This required TrkB kinase activity, a functional mitogen-activated protein kinase pathway, suppression of E-cadherin, and induction of Twist, a transcription factor contributing to EMT and metastasis. RNA interference (RNAi)-mediated Twist depletion blocked TrkB-induced EMT-like transformation, anoikis suppression, and growth of tumor xenografts. By searching for essential effectors of TrkB-Twist signaling, we found that Twist induces Snail, another EMT regulator associated with poor cancer prognosis. Snail depletion impaired EMT-like transformation and anoikis suppression induced by TrkB, but in contrast to Twist depletion, it failed to inhibit tumor growth. Instead, Snail RNAi specifically impaired the formation of lung metastases. Epistasis experiments suggested that Twist acts upstream from Snail. Our results demonstrate that TrkB signaling activates a Twist-Snail axis that is critically involved in EMT-like transformation, tumorigenesis, and metastasis. Moreover, our data shed more light on the epistatic relationship between Twist and Snail, two key transcriptional regulators of EMT and metastasis.

Autoregulation of Th1-mediated inflammation by twist1.

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor kappaB (NF-kappaB)-dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-kappaB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-gamma, IL-2, and tumor necrosis factor-alpha, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA.

BACKGROUND: The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. RESULTS: We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed) total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. CONCLUSION: We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.

Four twist genes in zebrafish, four expression patterns.

Twist genes code for regulatory bHLH proteins essential for embryonic development and conserved across the metazoa. There are four genes that constitute the zebrafish twist family: twist1a, twist1b, twist2--orthologs of the mammalian twist1 and twist2 genes; and twist3--a gene from a new clade that does not exist in mammals. Presented here are their embryonic mRNA expression profiles. The study extends the known conservation of twist developmental patterns in tetrapods to the fish, e.g., expression in cephalic neural crest, sclerotome and lateral plate mesoderm. Some other expression domains are unique, like hypochord and dorsal aorta; some, like the notochord, may be ancestral patterns retained from protochordates; and the expression in invaginating/migrating cells may have been retained from the jellyfish. Perhaps this is one of the more ancient functions of twist--conserved from diploblasts to humans--to facilitate cell movement.

Evolution of the vertebrate twist family and synfunctionalization: a mechanism for differential gene loss through merging of expression domains.

Twist genes are essential for embryonic development and are conserved from jellyfish to human. To study the vertebrate twist family and its evolution, the entire complement of twist genes was obtained for 9 representative species. Phylogenetic analysis showed that a single protochordate twist gene was duplicated at least twice before the teleost-tetrapod split to give rise to 3 ancestral genes, which were further duplicated or deleted, resulting in fluctuating number of twist paralogs in different vertebrate lineages. To find whether changes in gene copy number were associated with changes in gene function, embryonic expression patterns of twist orthologs were evaluated against the number of twist paralogs in different species. The results showed evidence for both neo- and subfunctionalization, and, in addition, for loss of an ancestral regulatory gene. For example, in Xenopus, twist2 was lost, but the twist1 paralog acquired, and therefore preserved, twist2 function. A general model is proposed to explain the data. In this process, termed synfunctionalization, one paralog acquires the expression domain(s) of another. The merging may lead to function shuffle. Alternatively, it may leave one paralog redundant and thus subject to deletion--while its function is retained by the surviving paralog(s). Synfunctionalization is a mechanism that, together with neo- and subfunctionalization, may work to establish equilibrium in the number of genes that regulate developmental processes; it may regulate the complexity of regulatory regions as well as gene copy number and therefore may play a role in evolution of gene function and the structure of genome.

Twist relates to tubular epithelial-mesenchymal transition and interstitial fibrogenesis in the obstructed kidney.

Epithelial-mesenchymal transition (EMT) is a critical step in renal fibrosis. It has been recently reported that a transcription factor, Twist, plays a pivotal role in metastasis of breast tumors by inducing EMT. In this study, we examined whether Twist relates to renal fibrogenesis including EMT of tubular epithelia, evaluating Twist expression level in the unilateral ureteral obstruction (UUO) model. Kidneys of mice subjected to UUO were harvested 1, 3, 7, and 10 days after obstruction. Compared with control kidneys, Twist mRNA-level significantly increased 3 days after UUO (UUO day 3 kidney) and further augmented until 10 days after UUO. Twist expression increased in tubular epithelia of the dilated tubules and the expanded interstitial areas of UUO kidneys, where cell-proliferating appearances were frequently found in a time-dependent manner. Although a part of tubular cells in whole nephron segment were immunopositive for Twist in UUO day 7 kidneys, tubular epithelia downstream of nephron more frequently expressed Twist than upstream of nephron. In UUO day 7 kidneys, some tubular epithelia were confirmed to coexpress Twist and fibroblast-specific protein-1, a marker for EMT, indicating that Twist is involved in tubular EMT under pathological state. Twist was expressed also in a number of alpha-smooth muscle actin-positive myofibroblasts located in the expanded interstitial area of UUO kidneys. From these findings, the present investigation suggests that Twist is associated with tubular EMT, proliferation of myofibroblasts, and subsequent renal fibrosis in obstructed kidneys.

A wide-range, low-cost 150 bp ladder for sizing DNA fragments between 150 and 4500 bp.

Agarose gel electrophoresis, a very routine procedure, requires molecular weight standards; these are usually manufactured from plasmid or viral DNA fragments, or more recently, from PCR products of defined sizes. We describe here the preparation of a molecular weight standard from a completely different DNA source - the uniquely organized genome of the beetle Tenebrio molitor. The standard can be used to accurately size DNAs between 150 and 4500 bp, a useful range of sizes for many agarose gel electrophoresis applications, including separation of PCR products and plasmid cloning targets. In addition, it is easy to prepare, inexpensive, and rivals the best of the commercial ladders.

Mesangial cells initiate compensatory renal tubular hypertrophy via IL-10-induced TGF-beta secretion: effect of the immunomodulator AS101 on this process.

The present study investigated the role of IL-10 produced by the mesangial cells in postnephrectomy compensatory renal growth and the effect of the immunomodulator AS101 on this process. One hundred forty unilateral nephrectomized and sham-operated male Sprague-Dawley rats were treated by AS101 or PBS before and after surgery. The results show that secretion of IL-10 and TGF-beta by mesangial cells isolated from the remaining kidneys was increased significantly, compared with those of control and sham animals. Moreover, TGF-beta secretion by mesangial cells was increased after the addition of exogenous recombinant IL-10 and inhibited in the presence of neutralizing anti-IL-10 antibodies. In vivo, compensatory growth of the remaining kidneys was associated with significant increase in IL-10 content in renal tissues and plasma. Immunohistochemical studies show that IL-10 was produced by mesangial cells. Elevated IL-10 levels were followed by the rise in TGF-beta content in plasma and renal tissue. AS101 treatment decreased IL-10 and TGF-beta expression in plasma and kidney tissues and results in 25% reduction in the fresh and fractional kidney weight and decreased hypertrophy of tubular cells (protein/DNA ratio, morphometric analysis). Taken together, these data demonstrate that TGF-beta production by mesangial cells is IL-10 dependent. Mesangial cells are the major source of IL-10 in kidneys. AS101, by inhibiting the activity of IL-10, decreases TGF-beta production by mesangial cells, thus limiting compensatory tubular cell hypertrophy.

Viral inoculation of mouse embryos in utero.

A technique is described for the injection of live virus into early- and mid-gestation mouse embryos in utero. The procedure is quick, easy, harmless to the embryos, and does not require specialized surgical or microinjection equipment. Since the developing embryo contains most different cell types in a very wide range of differentiation states, the procedure permits a rapid and near complete characterization of the host cell type range in a single experimental system. Under anaesthesia, a simple laparotomy was used to reveal the uterine horns of 9.5 or 12.5 days post-conception(dpc) females. One uterine horn was deflected onto the ventral abdominal surface. Embryos were injected through the uterine wall and the uterine horn replaced into the abdominal cavity. The entire operation could be completed in 10-15 min without distinguishable pain to the mother or adverse effect on the pregnancy. The procedure is presented in sufficient detail to permit its ready adoption in situations where a more complete characterization of host cell type range is sought.

Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis.

Metastasis is a multistep process during which cancer cells disseminate from the site of primary tumors and establish secondary tumors in distant organs. In a search for key regulators of metastasis in a murine breast tumor model, we have found that the transcription factor Twist, a master regulator of embryonic morphogenesis, plays an essential role in metastasis. Suppression of Twist expression in highly metastatic mammary carcinoma cells specifically inhibits their ability to metastasize from the mammary gland to the lung. Ectopic expression of Twist results in loss of E-cadherin-mediated cell-cell adhesion, activation of mesenchymal markers, and induction of cell motility, suggesting that Twist contributes to metastasis by promoting an epithelial-mesenchymal transition (EMT). In human breast cancers, high level of Twist expression is correlated with invasive lobular carcinoma, a highly infiltrating tumor type associated with loss of E-cadherin expression. These results establish a mechanistic link between Twist, EMT, and tumor metastasis.

A replacement for methoxyflurane (Metofane) in open-circuit anaesthesia.

Methoxyflurane (Metofane) has been widely used as an open-circuit anaesthetic in small laboratory animals for several decades. Its low vapour pressure and high blood solubility have permitted its use in convenient and simple drop-chamber/nose-cone setups. Recently, following the decision by the primary manufacturer to discontinue production, it has become increasingly difficult to obtain methoxyflurane. We describe here a simple and effective adaptation of isoflurane, an excellent inhalation anaesthetic, to open-circuit drop-chamber/nose-cone anaesthesia. It was found that the vapour concentration of isoflurane could be continuously varied by dissolving the anaesthetic in propylene glycol and that a 20% solution produced effective anaesthesia such that in adult mice, 2 ml of 20% isoflurane in propylene glycol induced anaesthesia within 2 min in a one-litre drop chamber. Furthermore, anaesthesia maintenance with 20% isoflurane was tested in two sets of mice. In one set, surgical plane anaesthesia was maintained for 10 min in a head chamber. After removal of the chamber, the animals awoke within one minute and recovered without any indication of post-anaesthetic distress. The second set contained pregnant mice; here anaesthesia was maintained for between 10 and 12 min, during which laparotomy, exposure of one uterine horn, intrauterine injection and wound closure were completed. The recovery from anaesthesia was also within a minute and with no signs of distress. Healthy litters were delivered after a normal gestation. This isoflurane/propylene glycol procedure is simple, effective and humane, and is a good substitute for methoxyflurane.

Msx2 and Twist cooperatively control the development of the neural crest-derived skeletogenic mesenchyme of the murine skull vault.

The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2 and TWIST function in the same or distinct pathways. We trace the origin of the calvarial foramen defect in Msx2 mutant mice to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. We show that this cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. We demonstrate, in addition, that heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2 and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level.

The P4 promoter of the parvovirus minute virus of mice is developmentally regulated in transgenic P4-LacZ mice.

Activation of the minute virus of mice (MVM) P4 promoter is a key step in the life cycle of the virus and is completely dependent on host transcription factors. Since transcription-factor composition varies widely in different cell types, there is the possibility that only some cell types in the host organism have the capacity to initiate expression from the P4 promoter and therefore that the promoter may be a factor in determining the tropism of MVM. In this study, the ability of various cell types to activate P4, independent of the other virus-host interactions, was examined in transgenic mouse lines bearing a beta-galactosidase reporter sequence driven by the P4 promoter. It was found that lacZ was expressed during embryogenesis and in the adult in a cell-type-specific and differentiation-dependent pattern. The data are consistent with cell-type and stage-specific activation of the P4 promoter having a role in determining the host cell-type range of MVM. The ability of some parvoviruses to replicate in, and kill oncogenically transformed cells, and to destroy induced tumors in laboratory animals is the basis of recent approaches to use MVM-based vectors in cancer gene therapy. Since these vectors rely on the activation of the P4 promoter by the target tissues, understanding the promoter dependence on cell-type and differentiation status is important for their design and potential use.

Preferential binding of Grb2 or phosphatidylinositol 3-kinase to the met receptor has opposite effects on HGF-induced myoblast proliferation.

Hepatocyte growth factor (HGF) and its receptor, Met, play a crucial role in regulating adult skeletal myoblast proliferation and differentiation. Met signaling is mediated by phosphorylation of two carboxy-terminal tyrosines, which act as docking sites for a number of intracellular mediators. These include Grb2 and p85, which couple the receptor with the Ras and phosphatidylinositol 3-kinase (PI3K) pathways, respectively. In this study, we define the role of these effectors in response to HGF by utilizing Met mutants, designed to obtain preferential coupling of Met to either Grb2 or PI3K or both. We found that relative to the wild-type receptor, enhanced binding to Grb2 further increases the incorporation of bromodeoxyuridine and the expression of Twist, while decreasing that of p27(Kip1) and myogenin. Conversely, preferential coupling with PI3K induced cell-cycle withdrawal and differentiation. Whereas enhanced Grb2 binding increased the phosphorylation of the mitogen-activated protein kinase/extracellular signal-regulated protein kinases (MAPK/ERK) and abrogated that of p38 MAPK, PI3K had the opposite effect. PD098059 reversed the inhibitory effects of Met on cell proliferation and differentiation, while wortmannin had only a very marginal effect. Taken together, these data suggest that coupling of Met with Grb2 is necessary for HGF-mediated inhibition of muscle differentiation. This inhibition occurs only when PI3K signaling downstream of Met is low. Imposing an efficient coupling of PI3K to Met would lead to upregulation of muscle regulatory factors and subsequent cell differentiation.