The Overexpression of IQGAP1 and β-Catenin Is Associated with Tumor Progression in Hepatocellular Carcinoma In Vitro and In Vivo

The IQ-domain GTPase-activating protein 1 (IQGAP1) is a multifunctional scaffold protein, which interacts with diverse proteins to regulate cell adhesion and cell migration. The abnormal expression of IQGAP1 widely exists in many cancers, but biological roles of IQGAP1 cooperation with its interacting proteins to involve in tumorigenesis remain to clarify. In this study, we have found that IQGAP1 interacts with β-catenin and regulates β-catenin expression in hepatocellular carcinoma (HCC) cells. The expression levels of IQGAP1 and β-catenin and their associations have a positive correlation with cell metastasis ability in several HCC cell lines. The up-regulation of IQGAP1 and β-catenin improves cell proliferation and migration ability of HCC cells, whereas the knockdown of IQGAP1 by small interfering RNA can decrease β-catenin expression, which results in the reduction of cell proliferation and migration ability in vitro. In addition, a significantly higher expression of IQGAP1 and β-catenin also usually exists in human HCC tissues, especially their overexpression is clinicopathologically associated with tumor malignancy. Generally the overexpression and interactions of IQGAP1 and β-catenin contribute to HCC progression by promoting cell proliferation and migration.     

Genetic Dissection of Differential Signaling Threshold Requirements for the Wnt/β-Catenin Pathway In Vivo

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/β-catenin pathway, we challenged the allele combinations by genetically restricting intracellular β-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/β-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/β-catenin signaling capacity similar to that in the germline of the Apc^min mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc^min mice arise independently of intestinal tumorigenesis. Together, the present genotype–phenotype analysis suggests tissue-specific response levels for the Wnt/β-catenin pathway that regulate both physiological and pathophysiological conditions.     

Role of MicroRNA Modulation in the Interferon-α/Ribavirin Suppression of HIV-1 In Vivo

Background

Interferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo.

Methods

Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors.

Results

miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo.

Conclusions

The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.     

Increased Cell Proliferation and Neurogenesis in the Hippocampal Dentate Gyrus of Old GFAP<Superscript>−/−</Superscript>Vim<Superscript>−/−</Superscript> Mice

In response to central nervous system (CNS) injury, and more discretely so also during aging, astrocytes become reactive and increase their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Studies of mice deficient in astrocytic intermediate filaments have provided insights into the function of reactive gliosis. Recently we demonstrated robust integrationof retinal transplants (1) and increased posttraumatic synaptic regeneration (2) in GFAP^–/–Vim^–/– mice, suggesting that modulation of astrocyte activity affects the permissiveness of the CNS environment for regeneration. Neurogenesis in the adult mammalian CNS is restricted to essentially two regions, the hippocampus and the subventricular zone. Here, we assessed neurogenesis in the hippocampus of 18-month-old GFAP^–/–Vim^–/– mice. In the granular layer of the dentate gyrus, cell proliferation/survival was 34% higher and neurogenesis 36% higher in GFAP^–/–Vim^–/– mice than in wildtype controls. These findings suggest that the adult hippocampal neurogenesis in healthy old mice can be increased by modulating astrocyte reactivity.Special issue dedicated to Lawrence. F. Eng.     

4′-Phosphopantetheinyl Transferase PptT,a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo

The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4′-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections.     

TDP2–Dependent Non-Homologous End-Joining Protects against Topoisomerase II–Induced DNA Breaks and Genome Instability in Cells and In Vivo

Anticancer topoisomerase “poisons” exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5′-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.     

Activation of AMP-Activated Protein Kinase by 3,3′-Diindolylmethane (DIM) Is Associated with Human Prostate Cancer Cell Death In Vitro and In Vivo

There is a large body of scientific evidence suggesting that 3,3′-Diindolylmethane (DIM), a compound derived from the digestion of indole-3-carbinol, which is abundant in cruciferous vegetables, harbors anti-tumor activity in vitro and in vivo. Accumulating evidence suggests that AMP-activated protein kinase (AMPK) plays an essential role in cellular energy homeostasis and tumor development and that targeting AMPK may be a promising therapeutic option for cancer treatment in the clinic. We previously reported that a formulated DIM (BR-DIM; hereafter referred as B-DIM) with higher bioavailability was able to induce apoptosis and inhibit cell growth, angiogenesis, and invasion of prostate cancer cells. However, the precise molecular mechanism(s) for the anti-cancer effects of B-DIM have not been fully elucidated. In the present study, we investigated whether AMP-activated protein kinase (AMPK) is a molecular target of B-DIM in human prostate cancer cells. Our results showed, for the first time, that B-DIM could activate the AMPK signaling pathway, associated with suppression of the mammalian target of rapamycin (mTOR), down-regulation of androgen receptor (AR) expression, and induction of apoptosis in both androgen-sensitive LNCaP and androgen-insensitive C4-2B prostate cancer cells. B-DIM also activates AMPK and down-regulates AR in androgen-independent C4-2B prostate tumor xenografts in SCID mice. These results suggest that B-DIM could be used as a potential anti-cancer agent in the clinic for prevention and/or treatment of prostate cancer regardless of androgen responsiveness, although functional AR may be required.     

Lipoprotein Lipase (LPL) is Associated with Neurite Pathology and Its Levels Are Markedly Reduced in the Dentate Gyrus of Alzheimer’s Disease Brains

Lipoprotein lipase (LPL) is involved in regulation of fatty acid metabolism, and facilitates cellular uptake of lipoproteins, lipids and lipid-soluble vitamins. We evaluated LPL distribution in healthy and Alzheimer’s disease (AD) brain tissue and its relative levels in cerebrospinal fluid. LPL immunostaining is widely present in different neuronal subgroups, microglia, astrocytes and oligodendroglia throughout cerebrum, cerebellum and spinal cord. LPL immunoreactivity is also present in leptomeninges, small blood vessels, choroid plexus and ependymal cells, Schwann cells associated with cranial nerves, and in anterior and posterior pituitary. In vitro studies have shown presence of secreted LPL in conditioned media of human cortical neuronal cell line (HCN2) and neuroblastoma cells (SK-N-SH), but not in media of cultured primary human astrocytes. LPL was present in cytoplasmic and nuclear fractions of neuronal cells and astrocytes in vitro. LPL immunoreactivity strongly associates with AD-related pathology, staining diffuse plaques, dystrophic and swollen neurites, possible Hirano bodies and activated glial cells. We observed no staining associated with neurofibrillary tangles or granulovacuolar degeneration. Granule cells of the dentate gyrus and the associated synaptic network showed significantly reduced staining in AD compared to control tissue. LPL was also reduced in AD CSF samples relative to those in controls.     

In Situ Immune Response in Human Chromoblastomycosis – A Possible Role for Regulatory and Th17 T Cells

Background

Chromoblastomycosis is a chronic fungal infection that affects skin and subcutaneous tissue. Lesions can be classified in tumorous, verrucous, cicatricial and plaque type. The cellular immune response in the severe form of the disease seems to correlate with a Th2 pattern of cytokines. The humoral immune response also seems to play a role. We intended to explore the populations of regulatory T cells and the Th17 pattern.

Methodology

Twenty-three biopsies of verrucous form were obtained from patients with clinical, culture and histopathological diagnostic of chromoblastomycosis, without treatment. It was performed an immunohistochemistry method to detect Foxp3, CD25, TGF-β, IL-6, IL-17 and IL-23.

Principal findings

IL-17 was the only cytokine with high expression in CBM when compared to normal skin. The expression of Treg cells, TGF- β, IL-6 and IL-23 were similar to normal skin.

Conclusions/Significance

The constitution of a local immune response with high expression of IL-17 and low expression of other cytokines could be at least in part, an attempt to help the immune system against fungal infection. On the other hand, high levels of local immune response mediated by Th17 profile could overcome the role of Treg cells. The inefficient immunomodulation as a consequence of the unbalance by Treg/Th17 cells seems to corroborate with the less effective immune response against fungi.     

Telomerase: A Target for Therapeutic Effects of Curcumin and a Curcumin Derivative in Aβ1-42 Insult In Vitro

This study was designed to investigate whether telomerase was involved in the neuroprotective effect of curcumin and Cur1. Alzheimer''s disease is a consequence of an imbalance between the generation and clearance of amyloid-beta peptide in the brain. In this study, we used Aβ1-42 (10 µg/ml) to establish a damaged cell model, and curcumin and Cur1 were used in treatment groups. We measured cell survival and cell growth, intracellular oxidative stress and hTERT expression. After RNA interference, the effects of curcumin and Cur1 on cells were verified. Exposure to Aβ1–42 resulted in significant oxidative stress and cell toxicity, and the expression of hTERT was significantly decreased. Curcumin and Cur1 both protected SK-N-SH cells from Aβ1–42 and up-regulated the expression of hTERT. Furthermore, Cur1 demonstrated stronger protective effects than curcumin. However, when telomerase was inhibited by TERT siRNA, the neuroprotection by curcumin and Cur1 were ceased. Our study indicated that the neuroprotective effects of curcumin and Cur1 depend on telomerase, and thus telomerase may be a target for therapeutic effects of curcumin and Cur1.     

Identification of the Lipopolysaccharide Core of Yersinia pestis and Yersinia pseudotuberculosis as the Receptor for Bacteriophage ��A1122

φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis.     

The peri-urban interface and house infestation with Triatoma infestans in the Argentine Chaco: an underreported process?

Peri-urban infestations with triatomine bugs, their sources and their dynamics haverarely been investigated. Here, we corroborated the reported occurrence ofTriatoma infestans in a peri-urban area and in neighbouring ruralhouses in Pampa del Indio, in the Argentine Chaco, and identified its putativesources using spatial analysis and demographic questionnaires. Peri-urbanhouseholders reported that 10% of their premises had triatomines, whereas T.infestans was collected by timed manual searches or community-basedsurveillance in only nine (3%) houses. Trypanosoma cruzi-infectedT. infestans and Triatoma sordida were collectedindoors only in peri-urban houses and were infected with TcVand TcI, respectively. The triatomines fed on chickens,cats and humans. Peri-urban infestations were most frequent in a squatter settlementand particularly within the recently built mud houses of rural immigrants, withlarge-sized households, more dogs and cats and more crowding. Several of the observedinfestations were most likely associated with passive bug transport from othersources and with active bug dispersal from neighbouring foci. Thus, the households inthe squatter settlement were at a greater risk of bug invasion and colonisation. Insum, the incipient process of domestic colonisation and transmission, along withpersistent rural-to-urban migratory flows and unplanned urbanisation, indicate theneed for active vector surveillance and control actions at the peri-urban interfaceof the Gran Chaco.     

Evidence for a “Wattle and Daub” Model of the Cyst Wall of Entamoeba

The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a “wattle and daub” model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).     

What Is the Optimal Value of the g-Ratio for Myelinated Fibers in the Rat CNS? A Theoretical Approach

Background

The biological process underlying axonal myelination is complex and often prone to injury and disease. The ratio of the inner axonal diameter to the total outer diameter or g-ratio is widely utilized as a functional and structural index of optimal axonal myelination. Based on the speed of fiber conduction, Rushton was the first to derive a theoretical estimate of the optimal g-ratio of 0.6 [1]. This theoretical limit nicely explains the experimental data for myelinated axons obtained for some peripheral fibers but appears significantly lower than that found for CNS fibers. This is, however, hardly surprising given that in the CNS, axonal myelination must achieve multiple goals including reducing conduction delays, promoting conduction fidelity, lowering energy costs, and saving space.

Methodology/Principal Findings

In this study we explore the notion that a balanced set-point can be achieved at a functional level as the micro-structure of individual axons becomes optimized, particularly for the central system where axons tend to be smaller and their myelin sheath thinner. We used an intuitive yet novel theoretical approach based on the fundamental biophysical properties describing axonal structure and function to show that an optimal g-ratio can be defined for the central nervous system (≈0.77). Furthermore, by reducing the influence of volume constraints on structural design by about 40%, this approach can also predict the g-ratio observed in some peripheral fibers (≈0.6).

Conclusions/Significance

These results support the notion of optimization theory in nervous system design and construction and may also help explain why the central and peripheral systems have evolved different g-ratios as a result of volume constraints.     

Siderophore uptake in bacteria and the battle for iron with the host; a bird’s eye view

Siderophores are biosynthetically produced and secreted by many bacteria, yeasts, fungi and plants, to scavenge for ferric iron (Fe³⁺). They are selective iron-chelators that have an extremely high affinity for binding this trivalent metal ion. The ferric ion is poorly soluble but it is the form of iron that is predominantly found in oxygenated environments. Siderophore uptake in bacteria has been extensively studied and over the last decade, detailed structural information for many of the proteins that are involved in their transport has become available. Specifically, numerous crystal structures for outer membrane siderophore transporters, as well as for soluble periplasmic siderophore-binding proteins, have been reported. Moreover, unique siderophore-binding proteins have recently been serendipitously discovered in humans, and the structures of some of their siderophore-complexes have been characterized. The binding pockets for different ferric-siderophores in these proteins have been described in great molecular detail. In addition to highlighting this structural information, in this review paper we will also briefly discuss the relevant chemical properties of iron, and provide a perspective on our current understanding of the human and bacterial iron uptake pathways. Potential clinical uses of siderophores will also be discussed. The emerging overall picture is that iron metabolism plays an extremely important role during bacterial infections. Because levels of free ferric iron in biological systems are always extremely low, there is serious competition for iron and for ferric-siderophores between pathogenic bacteria and the human or animal host.