Insertional variability of four transposable elements and population structure of the midge Chironomus riparius (Diptera)

The dipteran Chironomus riparius is found across the entire Palearctic region; its larvae are among the most abundant macroinvertebrates inhabiting inland waterbodies. Chironomid larvae have been extensively used in ecotoxicological and cytogenetic research, but relatively little is known on the population structure of this species. Transposable elements (TEs) are DNA sequences that are capable of autonomous replication; the number and genomic location of TE insertions varies across individuals; this variability is increasingly being used in population studies. Several TEs had been characterized in Chironomids; this enabled the analysis of insertional variability of four different TEs in six natural populations of C. riparius from Italy, Bulgaria and Russia using a PCR-based method, transposon insertion display (TID). The method allows to obtain dominant markers, similar to AFLP. In all populations, TE insertions showed high individual polymorphism, while median copy numbers of the same TEs did not vary between populations. Analysis of molecular variance (AMOVA) detected significant differentiation between populations for three of the TEs; although no correlation between genetic and geographic distances was found, the corresponding population structures were found to be significantly correlated and indicate a degree of isolation by distance. TEs belonging to different classes have different mechanisms of replication, resulting in different transposition rates of mobilization; the finding of mostly concordant population structuring for three of the TEs indicates that population dynamics contributed significantly in shaping the detected insertional polymorphism.     

Happy aged people are all alike, while every unhappy aged person is unhappy in its own way

Aging of the world's population represents one of the most remarkable success stories of medicine and of humankind, but it is also a source of various challenges. The aim of the collaborative cross-cultural European study of adult well being (ESAW) is to frame the concept of aging successfully within a causal model that embraces physical health and functional status, cognitive efficacy, material security, social support resources, and life activity. Within the framework of this project, we show here that the degree of heterogeneity among people who view aging in a positive light is significantly lower than the degree of heterogeneity of those who hold a negative perception of aging. We base this conclusion on our analysis of a survey involving 12,478 people aged 50 to 90 from six West European countries. We treat the survey database as a bipartite network in which individual respondents are linked to the actual answers they provide. Taking this perspective allows us to construct a projected network of respondents in which each link indicates a statistically validated similarity of answers profile between the connected respondents, and to identify clusters of individuals independently of demographics. We show that mental and physical well-being are key factors determining a positive perception of aging. We further observe that psychological aspects, like self-esteem and resilience, and the nationality of respondents are relevant aspects to discriminate among participants who indicate positive perception of aging.     

Development of Buffalo Hump in the course of antiretroviral therapy including raltegravir and unboosted atazanavir: a case report and review of the literature

Introduction

The availability of raltegravir plus atazanavir provides an alternative antiretroviral strategy that may be equally efficacious and less toxic than those currently recommended in HIV treatment guidelines. In fact, this new combination antiretroviral therapy attracts the attention of the scientific community because both drugs have a good safety profile coupled with potent antiviral activity, and their combined use would avert nucleoside- and ritonavir-related toxicities.

Case presentation

We describe the case of a 47-year-old, Caucasian woman treated for HIV-1 infection who developed Buffalo Hump during antiretroviral therapy, including raltegravir and unboosted atazanavir. Clinical evaluation and an ultrasonography scan of the cervical region showed a new progressive increase of lipohypertrophy and the results of DEXA confirmed these data. In our patient the worsening of the Buffalo Hump cannot be attributed to hypercortisolism; insulin-resistance, diabetes, dyslipidemia, hyperlactatemia and metabolic syndrome were not present. Moreover, she was not in therapy with antiretroviral drugs that are described as the cause of Buffalo Hump; on the other hand she developed this side effect three months after the switch of the antiretroviral therapy to raltegravir plus unboosted atazanavir.

Conclusion

Current data indicate that the etiology of HIV-associated Buffalo Hump remains elusive but is likely multifactorial; a possible contributing cause, but not the main cause, could be exposure to antiretroviral drugs. To the best of our knowledge, this is the first report on development of Buffalo Hump in the course of antiretroviral therapy, including the use of these drugs. On the basis of our data we can formulate the hypothesis of a pharmacological pathogenesis that underlies the development of this case of Buffalo Hump in the absence of other risk factors.

     

A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro

Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.     

A functionally characterized test set of human induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.     

Evaluation of Trichoderma harzianum strain T22 as biological control agent of Calonectria pauciramosa

The objective of this research was to evaluate Trichoderma harzianum strain T22 as a biocontrol agent of collar and root rot caused by different Calonectria pauciramosa isolates. Thus, the microsclerotia-forming ability and virulence of twenty C. pauciramosa isolates were assessed. Microsclerotia production varied partially among the isolates and dual culture with T22 on carnation leaf agar revealed isolates with both high and low microsclerotia-forming ability. Inoculation tests on red clover (Triflolium pratense) demonstrated its susceptibility to the pathogen. On red clover, the degree of virulence and T22 effects in controlling infections were highly variable among the isolates tested. A nursery trial performed on Feijoa sellowiana seedlings confirmed previous results, clearly indicating virulence variability among C. pauciramosa isolates. For three isolates tested in nursery trial, T22 effectiveness in controlling infection was inversely related to their degree of virulence. Overall, T. harzianum strain T22 showed good antagonist activity in reducing microsclerotia production on carnation leaf and the incidence and severity of collar and root rot on both selected hosts. This data could be crucial in developing integrated pest management strategies in ornamental plant nurseries.     

The vascular endothelial growth factor (VEGF)-E encoded by orf virus regulates keratinocyte proliferation and migration and promotes epidermal regeneration

Vascular endothelial growth factor (VEGF)-A, a key regulator of cutaneous blood vessel formation, appears to have an additional role during wound healing, enhancing re-epithelialization. Orf virus, a zoonotic parapoxvirus, induces proliferative skin lesions that initiate in wounds and are characterized by extensive blood vessel formation, epidermal hyperplasia and rete ridge formation. The vascular changes beneath the lesion are largely due to viral-expressed VEGF-E. This study investigated using mouse skin models whether VEGF-E can induce epidermal changes such as that seen in the viral lesion. Injection of VEGF-E into normal skin increased the number of endothelial cells and blood vessels within the dermis and increased epidermal thickening and keratinocyte number. Injection of VEGF-E into wounded skin, which more closely mimics orf virus lesions, increased neo-epidermal thickness and area, promoted rete ridge formation, and enhanced wound re-epithelialization. Quantitative RT-PCR analysis showed that VEGF-E did not induce expression of epidermal-specific growth factors within the wound, but did increase matrix metalloproteinase (MMP)-2 and MMP-9 expression. In cell-based assays, VEGF-E induced keratinocyte migration and proliferation, responses that were inhibited by a neutralizing antibody against VEGF receptor (VEGFR)-2. These findings demonstrate that VEGF-E, both directly and indirectly, regulates keratinocyte function, thereby promoting epidermal regeneration.     

Adaptation of mouse skeletal muscle to long-term microgravity in the MDS mission

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5-20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca(2+)-activated K(+) channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.     

Contribution by polymorphonucleate granulocytes to elevated gamma-glutamyltransferase in cystic fibrosis sputum

Background

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gamma-glutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail.

Principal Findings

The present study was aimed to evaluate the origin and the biochemical characteristics of the GGT detectable in CF sputum. We found GGT activity both in neutrophils and in the fluid, the latter significantly correlating with myeloperoxidase expression. In neutrophils, GGT was associated with intracellular granules. In the fluid, gel-filtration chromatography showed the presence of two distinct GGT fractions, the first corresponding to the human plasma b-GGT fraction, the other to the free enzyme. The same fractions were also observed in the supernatant of ionomycin and fMLP-activated neutrophils. Western blot analysis confirmed the presence of a single band of GGT immunoreactive peptide in the CF sputum samples and in isolated neutrophils.

Conclusions

In conclusion, our data indicate that neutrophils are able to transport and release GGT, thus increasing GGT activity in CF sputum. The prompt release of GGT may have consequences on all GGT substrates, including major inflammatory mediators such as S-nitrosoglutathione and leukotrienes, and could participate in early modulation of inflammatory response.