MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3′ untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.     

Rescue of Amyloid-Beta-Induced Inhibition of Nicotinic Acetylcholine Receptors by a Peptide Homologous to the Nicotine Binding Domain of the Alpha 7 Subtype

Alzheimer''s disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.     

Distribution of Spotted Fever Group Rickettsiae in Hard Ticks (Ixodida: Ixodidae) from Panamanian Urban and Rural Environments (2007–2013)

Tick-borne rickettsiosis is an important emerging disease in Panama; to date, there have been 12 confirmed cases, including eight fatalities. To evaluate the distribution of rickettsiae in Panamanian ticks, we collected questing and on-host ticks in urban and rural towns in elevations varying between 0 and 2300 m. A total of 63 sites (13 urban and 50 rural towns) were used to develop models of spatial distributions. We found the following tick species: Rhipicephalus sanguineus s.l. (present in 54 of 63 towns and cities), Amblyomma mixtum (45/63), Dermacentor nitens (40/63), A. ovale (37/63), Rhipicephalus microplus (33/63), A. oblongoguttatum (33/63), Ixodes affinis (3/63), and Ixodes boliviensis (2/63). Rhipicephalus sanguineus s.l. was present in urban and rural towns, and other species were present only in rural towns. DNA was extracted from 408 R. sanguineus s.l., 387 A. mixtum, 103 A. ovale, and 11 A. oblongoguttatum and later tested for rickettsiae genes using PCR. Rickettsia DNA was detected in ticks from 21 of 63 localities. Rickettsia rickettsii was detected in five A. mixtum (1.29%), and Candidatus “Rickettsia amblyommii” was found in 138 A. mixtum (35%), 14 R. sanguineus (3.4%), and one A. ovale (0.9%). These results suggest that much of rural Panama is suitable for the expansion of tick populations and could favor the appearance of new tick-borne rickettsiosis outbreaks.     

Evaluation of dental enamel caries assessment using Quantitative Light Induced Fluorescence and Optical Coherence Tomography

An in vitro study of morphological alterations between sound dental structure and artificially induced white spot lesions in human teeth, was performed through the loss of fluorescence by Quantitative Light‐Induced Fluorescence (QLF) and the alterations of the light attenuation coefficient by Optical Coherence Tomography (OCT). To analyze the OCT images using a commercially available system, a special algorithm was applied, whereas the QLF images were analyzed using the software available in the commercial system employed. When analyzing the sound region against white spot lesions region by QLF, a reduction in the fluorescence intensity was observed, whilst an increase of light attenuation by the OCT system occurred. Comparison of the percentage of alteration between optical properties of sound and artificial enamel caries regions showed that OCT processed images through the attenuation of light enhanced the tooth optical alterations more than fluorescence detected by QLF System.

QLF versus OCT imaging of enamel caries: a photonics assessment     

Autophagy proteins are not universally required for phagosome maturation

Phagocytosis plays a central role in immunity and tissue homeostasis. After internalization of cargo into single-membrane phagosomes, these compartments undergo a maturation sequences that terminates in lysosome fusion and cargo degradation. Components of the autophagy pathway have recently been linked to phagosome maturation in a process called LC3-associated phagocytosis (LAP). In this process, autophagy machinery is thought to conjugate LC3 directly onto the phagosomal membrane to promote lysosome fusion. However, a recent study has suggested that ATG proteins may in fact impair phagosome maturation to promote antigen presentation. Here, we examined the impact of ATG proteins on phagosome maturation in murine cells using FCGR2A/FcγR-dependent phagocytosis as a model. We show that phagosome maturation is not affected in Atg5-deficient mouse embryonic fibroblasts, or in Atg5- or Atg7-deficient bone marrow-derived macrophages using standard assays of phagosome maturation. We propose that ATG proteins may be required for phagosome maturation under some conditions, but are not universally required for this process.     

The oceanic concordance of phylogeography and biogeography: a case study in Notochthamalus

Dispersal and adaptation are the two primary mechanisms that set the range distributions for a population or species. As such, understanding how these mechanisms interact in marine organisms in particular – with capacity for long‐range dispersal and a poor understanding of what selective environments species are responding to – can provide useful insights for the exploration of biogeographic patterns. Previously, the barnacle Notochthamalus scabrosus has revealed two evolutionarily distinct lineages with a joint distribution that suggests an association with one of the two major biogeographic boundaries (~30°S) along the coast of Chile. However, spatial and genomic sampling of this system has been limited until now. We hypothesized that given the strong oceanographic and environmental shifts associated with the other major biogeographic boundary (~42°S) for Chilean coastal invertebrates, the southern mitochondrial lineage would dominate or go to fixation in locations further to the south. We also evaluated nuclear polymorphism data from 130 single nucleotide polymorphisms to evaluate the concordance of the signal from the nuclear genome with that of the mitochondrial sample. Through the application of standard population genetic approaches along with a Lagrangian ocean connectivity model, we describe the codistribution of these lineages through a simultaneous evaluation of coastal lineage frequencies, an approximation of larval behavior, and current‐driven dispersal. Our results show that this pattern could not persist without the two lineages having distinct environmental optima. We suggest that a more thorough integration of larval dynamics, explicit dispersal models, and near‐shore environmental analysis can explain much of the coastal biogeography of Chile.     

CCR2 and CCR5 genes polymorphisms in women with cervical lesions from Pernambuco,Northeast Region of Brazil: a case-control study

Polymorphisms in chemokine receptors play an important role in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer (CC). Our study examined the association of CCR2-64I (rs1799864) andCCR5-Δ32 (rs333) polymorphisms with susceptibility to develop cervical lesion (CIN and CC) in a Brazilian population. The genotyping of 139 women with cervical lesions and 151 women without cervical lesions for the CCR2-64I and CCR5-Δ32 polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism. The individuals carrying heterozygous or homozygous genotypes (GA+AA) for CCR2-64I polymorphisms seem to be at lower risk for cervical lesion [odds ratio (OR) = 0.37, p = 0.0008)]. The same was observed for the A allele (OR = 0.39, p = 0.0002), while no association was detected (p > 0.05) with CCR5-Δ32 polymorphism. Regarding the human papillomavirus (HPV) type, patients carrying the CCR2-64Ipolymorphism were protected against infection by HPV type 16 (OR = 0.35, p = 0.0184). In summary, our study showed a protective effect ofCCR2-64I rs1799864 polymorphism against the development of cervical lesions (CIN and CC) and in the susceptibility of HPV 16 infection.