MiR-7 Triggers Cell Cycle Arrest at the G1/S Transition by Targeting Multiple Genes Including Skp2 and Psme3

MiR-7 acts as a tumour suppressor in many cancers and abrogates proliferation of CHO cells in culture. In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27^KIP and temporary growth arrest of CHO cells in the G1 phase. Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis. This work provides further mechanistic insight into the role of miR-7 as a regulator of cell growth in times of cellular stress.     

Autophagy and Exosomes in the Aged Retinal Pigment Epithelium: Possible Relevance to Drusen Formation and Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch''s membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch''s membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly.     

Behavior of geladas and other endemic wildlife during a desert locust outbreak at Guassa,Ethiopia: ecological and conservation implications

Desert locust (Schistocerca gregaria) outbreaks have occurred repeatedly throughout recorded history in the Horn of Africa region, devastating crops and contributing to famines. In June 2009, a desert locust swarm invaded the Guassa Plateau, Ethiopia, a large and unusually intact Afroalpine tall-grass ecosystem, home to important populations of geladas (Theropithecus gelada), Ethiopian wolves (Canis simensis), thick-billed ravens (Corvus crassirostris), and other Ethiopian or Horn of Africa endemics. During the outbreak and its aftermath, we observed many animals, including geladas, ravens, and a wolf, feeding on locusts in large quantities. These observations suggest surprising flexibility in the normally highly specialized diets of geladas and wolves, including the potential for temporary but intensive insectivory during locust outbreaks. To our knowledge, Guassa is the highest elevation site (3,200–3,600 m) at which desert locusts, which require temperatures >20°C for sustained flight, have been reported. Continued monitoring will be necessary to determine whether the June 2009 outbreak was an isolated incident or part of an emerging pattern in the Ethiopian Highlands linked to global warming. The intensive consumption of desert locusts by geladas, wolves, and ravens during the outbreak at Guassa raises concerns about pesticide-based locust control strategies and potential unintended adverse effects on endemic and endangered wildlife.     

Hilar GABAergic interneuron activity controls spatial learning and memory retrieval

Background

Although extensive research has demonstrated the importance of excitatory granule neurons in the dentate gyrus of the hippocampus in normal learning and memory and in the pathogenesis of amnesia in Alzheimer''s disease (AD), the role of hilar GABAergic inhibitory interneurons, which control the granule neuron activity, remains unclear.

Methodology and Principal Findings

We explored the function of hilar GABAergic interneurons in spatial learning and memory by inhibiting their activity through Cre-dependent viral expression of enhanced halorhodopsin (eNpHR3.0)—a light-driven chloride pump. Hilar GABAergic interneuron-specific expression of eNpHR3.0 was achieved by bilaterally injecting adeno-associated virus containing a double-floxed inverted open-reading frame encoding eNpHR3.0 into the hilus of the dentate gyrus of mice expressing Cre recombinase under the control of an enhancer specific for GABAergic interneurons. In vitro and in vivo illumination with a yellow laser elicited inhibition of hilar GABAergic interneurons and consequent activation of dentate granule neurons, without affecting pyramidal neurons in the CA3 and CA1 regions of the hippocampus. We found that optogenetic inhibition of hilar GABAergic interneuron activity impaired spatial learning and memory retrieval, without affecting memory retention, as determined in the Morris water maze test. Importantly, optogenetic inhibition of hilar GABAergic interneuron activity did not alter short-term working memory, motor coordination, or exploratory activity.

Conclusions and Significance

Our findings establish a critical role for hilar GABAergic interneuron activity in controlling spatial learning and memory retrieval and provide evidence for the potential contribution of GABAergic interneuron impairment to the pathogenesis of amnesia in AD.     

Granulin-epithelin precursor is an oncofetal protein defining hepatic cancer stem cells

Background and Aims

Increasing evidence has suggested that hepatocellular carcinoma (HCC) might originate from a distinct subpopulation called cancer stem cells (CSCs), which are responsible for the limited efficacy of conventional therapies. We have previously demonstrated that granulin-epithelin precursor (GEP), a pluripotent growth factor, is upregulated in HCC but not in the adjacent non-tumor, and that GEP is a potential therapeutic target for HCC. Here, we characterized its expression pattern and stem cell properties in fetal and cancerous livers.

Methods

Protein expression of GEP in fetal and adult livers was examined in human and mouse models by immunohistochemical staining and flow cytometry. Liver cancer cell lines, isolated based on their GEP and/or ATP-dependent binding cassette (ABC) drug transporter ABCB5 expression, were evaluated for hepatic CSC properties in terms of colony formation, chemoresistance and tumorigenicity.

Results

We demonstrated that GEP was a hepatic oncofetal protein that expressed in the fetal livers, but not in the normal adult livers. Importantly, GEP+ fetal liver cells co-expressed the embryonic stem (ES) cell-related signaling molecules including β-catenin, Oct4, Nanog, Sox2 and DLK1, and also hepatic CSC-markers CD133, EpCAM and ABCB5. Phenotypic characterization in HCC clinical specimens and cell lines revealed that GEP+ cancer cells co-expressed these stem cell markers similarly as the GEP+ fetal liver cells. Furthermore, GEP was shown to regulate the expression of ES cell-related signaling molecules β-catenin, Oct4, Nanog, and Sox2. Isolated GEP^high cancer cells showed enhanced colony formation ability and chemoresistance when compared with the GEP_low counterparts. Co-expression of GEP and ABCB5 better defined the CSC populations with enhanced tumorigenic ability in immunocompromised mice.

Conclusions

Our findings demonstrate that GEP is a hepatic oncofetal protein regulating ES cell-related signaling molecules. Co-expression of GEP and ABCB5 further enriches a subpopulation with enhanced CSC properties. The current data provide new insight into the therapeutic strategy.     

Spiroquinazolinones as novel, potent, and selective PDE7 inhibitors. Part 2: Optimization of 5,8-disubstituted derivatives

The optimization of 5,8-disubstituted spirocyclohexane-quinazolinones into potent, selective, soluble PDE7 inhibitors with acceptable in vivo pharmacokinetic parameters is presented.     

Ultrastructural characterization of whole hydrosalpinx from infertile Chinese women

Hydrosalpinx (HSP) has been shown to be detrimental to the outcome of assisted reproduction, but little is known of its pathology. This prospective study examined and detailed ultrastructural characterization of HSP of infertile women presenting for assisted reproductive treatments. Both light and electron microscopies were used to characterize HSP. Hematoxylin and eosin staining of HSP showed areas without epithelial cell lining or with abnormalities such as flattening of the epithelial layer and exfoliation of epithelial cells with occasional normal columnar epithelial lining. HSP muscle fibers were atrophic and occasionally replaced by fibrous tissues, or separated by areas of severe edema. Inflammatory cells could be found in hydrosalpinx fluid (HF) in the lumen in areas with flattened to no epithelial cells, without epithelial lining, as well as in dilated blood vessels and/or lymph vessels. Scanning electron microscopy of the epithelial surface revealed epithelial denudation-severe loss of both cilia and microvilli and stomata exuding globular bodies on eroded ampulla surfaces. Severe chronic inflammation and damage to the epithelial lining and musculature of Fallopian tubes and the presence of inflammatory cells provides an explanation for HF formation, and thus for the detrimental effects of HF on reproductive processes and IVF outcome.     

Aggressive amyloidosis in mice expressing human amyloid peptides with the Arctic mutation

The Arctic mutation within the amyloid-beta (Abeta) peptide causes Alzheimer disease. In vitro, Arctic-mutant Abeta forms (proto)fibrils more effectively than wild-type Abeta. We generated transgenic mouse lines expressing Arctic-mutant human amyloid precursor proteins (hAPP). Amyloid plaques formed faster and were more extensive in Arctic mice than in hAPP mice expressing wild-type Abeta, even though Arctic mice had lower Abeta(1-42/1-40) ratios. Thus, the Arctic mutation is highly amyloidogenic in vivo.     

Cyclic enkephalin-deltorphin hybrids containing a carbonyl bridge: structure and opioid activity

Six hybrid N-ureidoethylamides of octapeptides in which an N-terminal cyclic structure related to enkephalin was elongated by a C-terminal fragment of deltorphin were synthesized on MBHA resin. The synthetic procedure involved deprotection of Boc groups with HCl/dioxane and cleavage of the peptide resin with 45 % TFA in DCM. d-Lys and d-Orn were incorporated in position 2, and Lys, Orn, Dab, or Dap in position 5. The side chains of the dibasic amino function were protected with the Fmoc group. This protection was removed by treatment with 55 % piperidine in DMF, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate. Using various combinations of dibasic amino acids, peptides containing a 17-, 18-, 19- or 20-membered ring structure were obtained. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid activities were observed, depending on the size of the ring. Extension of the enkephalin sequence at the C-terminus by a deltorphin fragment resulted in a change of receptor selectivity in favor of the δ receptor. The conformational propensities of selected peptides were determined using the EDMC method in conjunction with data derived from NMR experiments carried out in water. This approach allowed proper examination of the dynamical behavior of these small peptides. The results were compared with those obtained earlier with corresponding N-(ureidoethyl)pentapeptide amides.     

Identification of Immunogenic Salmonella enterica Serotype Typhi Antigens Expressed in Chronic Biliary Carriers of S. Typhi in Kathmandu,Nepal

Background

Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage.

Methodology/Principal Findings

To study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT), to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479), an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70%) chronic carriers, 0 of 8 bile culture-negative controls (0%), 0 of 8 healthy Bangladeshis (0%), and 1 of 8 (12.5%) Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present.

Conclusions/Significance

Further evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic S. Typhi carriers.