Transient proteasome inhibition as a strategy to enhance lentiviral transduction of hematopoietic CD34(+) cells and T lymphocytes: implications for the use of low viral doses and large-size vectors

The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.8% vs 10.7%, p < 0.0001). Treatment with MG132 (0.5 μM) retained its beneficial effect with 3 different LV of increasing size up to 10.9 Kb (p < 0.01). We extended, for the first time, the application of proteasome inhibition to the transduction of T lymphocytes. A transient exposure to MG132 significantly improved lentiviral T-cell transduction. The mean percentage of transduced T cells progressively increased from 13.5% of untreated cells, to 21% (p = 0.3), 30% (p = 0.03) and 37% (p = 0.01) of T lymphocytes that were pre-treated with MG132 at 0.1, 0.5 and 1 μM, respectively. MG132 did not affect viability or functionality of HSC or T cells, nor significantly increased the number of integrated vector copies. Transient proteasome inhibition appears as a new procedure to safely enhance lentiviral transduction of HSC and T lymphocytes with low viral doses. This approach could be useful in settings where the use of large size vectors may impair optimal viral production.     

Segregation of virulent influenza A(H1N1) variants in the lower respiratory tract of critically ill patients during the 2010-2011 seasonal epidemic

Background

Since its appearance in 2009, the pandemic influenza A(H1N1) virus circulated worldwide causing several severe infections.

Methods

Respiratory samples from patients with 2009 influenza A(H1N1) and acute respiratory distress attending 24 intensive care units (ICUs) as well as from patients with lower respiratory tract infections not requiring ICU admission and community upper respiratory tract infections in the Lombardy region (10 million inhabitants) of Italy during the 2010–2011 winter-spring season, were analyzed.

Results

In patients with severe ILI, the viral load was higher in bronchoalveolar lavage (BAL) with respect to nasal swab (NS), (p<0.001) suggesting a higher virus replication in the lower respiratory tract. Four distinct virus clusters (referred to as cluster A to D) circulated simultaneously. Most (72.7%, n = 48) of the 66 patients infected with viruses belonging to cluster A had a severe (n = 26) or moderate ILI (n = 22). Amino acid mutations (V26I, I116M, A186T, D187Y, D222G/N, M257I, S263F, I286L/M, and N473D) were observed only in patients with severe ILI. D222G/N variants were detected exclusively in BAL samples.

Conclusions

Multiple virus clusters co-circulated during the 2010–2011 winter-spring season. Severe or moderate ILI were associated with specific 2009 influenza A(H1N1) variants, which replicated preferentially in the lower respiratory tract.     

Employing mutants to study thrombin residues responsible for factor XIII activation peptide recognition: a kinetic study

In the last stages of coagulation, thrombin helps to activate Factor XIII. The resultant transglutaminase introduces covalent cross-links into fibrin thus promoting clot stability. To better understand the roles of individual thrombin residues in recognition and hydrolysis of the Factor XIII activation peptide, mutations within thrombin's aryl and apolar binding site were explored. The thrombin mutants W215A, E217A, W215A/E217A, L99A, and I174A were examined through HPLC kinetics against the substrates FXIII (28-41) V34 AP and FXIII (28-41) V34L AP. Several mutants responded differently to FXIII (28-41) V34 AP vs the cardioprotective V34L AP. W215 provides an important platform for binding and directing FXIII APs for proper hydrolysis. Loss of this platform leads to decreases in kinetics, particularly to the kcat of FXIII V34L AP. E217 also plays a supporting role, but the E217A mutation is not as detrimental as W215A. W215A/E217A is unfavorable for both activation peptides and its coupling effect has been characterized. This mutant can readily bind the peptides but cannot orient them for effective hydrolysis. Kinetic studies with I174A indicate that this thrombin residue is more crucial for interactions with the larger V34L AP segment. The L99A mutation causes deleterious effects to binding and hydrolysis of both APs. The V34L, however, is able to partially compensate for the loss perhaps by increasing contact within the aryl and apolar sites. Understanding how specific FXIII and thrombin residues participate in binding and control hydrolysis may lead to the design of coagulation enzymes whose degree of activation and optimal target site can be controlled.     

Trehalose inhibits cell proliferation and amplifies long-term temozolomide- and radiation-induced cytotoxicity in melanoma cells: A role for autophagy and premature senescence

Cutaneous melanomas frequently metastasize to the brain, with temozolomide (TMZ) plus radiotherapy (RT) offering little control of these lesions. We tested whether trehalose, a natural glucose disaccharide proved to induce autophagy, could enhance the effect of TMZ and ionizing radiation (IR). In two melanoma cell lines (A375 and SK-Mel-28), which greatly differ in chemosensitivity and radiosensitivity, trehalose significantly inhibited short-term cell proliferation and also enhanced IR-induced cytostasis. Interestingly, in TMZ-resistant SK-Mel-28 cells, trehalose was more effective than TMZ, and combined trehalose + TMZ further reduced cell proliferation. In long-term experiments, colony-forming capacity was dramatically reduced by trehalose, and even more by combined trehalose + TMZ or trehalose + IR. In resistant SK-Mel-28 cells, although growth was inhibited most with trehalose + TMZ + IR-6 Gy combined treatment, it is notable that trehalose + TMZ treatment was also very effective. Along with a direct antiproliferative effect, two further mechanisms may explain how trehalose potentiates TMZ- and IR-induced effects: the remarkable trehalose-stimulated autophagy in A375 cells, which were sensitive to TMZ- and IR-induced apoptosis; and the notable trehalose-stimulated premature senescence in SK-Mel-28 cells, which were resistant to apoptosis and less prone to autophagy. In normal melanocytes, trehalose induced a minor autophagy and cell proliferation inhibition, without affecting cell viability; moreover, when trehalose was used in combination with TMZ, the slight TMZ-induced cytotoxicity was not significantly reinforced. Together, our results suggest that trehalose, a safe nutrient supplement able to cross the blood–brain barrier, is a promising candidate, worthy to be further explored in vivo, to augment the therapeutic efficacy of TMZ and RT in melanoma brain metastases.     

Insight into the roles of selection in speciation from genomic patterns of divergence and introgression in secondary contact in venomous rattlesnakes

Investigating secondary contact of historically isolated lineages can provide insight into how selection and drift influence genomic divergence and admixture. Here, we studied the genomic landscape of divergence and introgression following secondary contact between lineages of the Western Diamondback Rattlesnake (Crotalus atrox) to determine whether genomic regions under selection in allopatry also contribute to reproductive isolation during introgression. We used thousands of nuclear loci to study genomic differentiation between two lineages that have experienced recent secondary contact following isolation, and incorporated sampling from a zone of secondary contact to identify loci that are resistant to gene flow in hybrids. Comparisons of patterns of divergence and introgression revealed a positive relationship between allelic differentiation and resistance to introgression across the genome, and greater‐than‐expected overlap between genes linked to lineage‐specific divergence and loci that resist introgression. Genes linked to putatively selected markers were related to prominent aspects of rattlesnake biology that differ between populations of Western Diamondback rattlesnakes (i.e., venom and reproductive phenotypes). We also found evidence for selection against introgression of genes that may contribute to cytonuclear incompatibility, consistent with previously observed biased patterns of nuclear and mitochondrial alleles suggestive of partial reproductive isolation due to cytonuclear incompatibilities. Our results provide a genome‐scale perspective on the relationships between divergence and introgression in secondary contact that is relevant for understanding the roles of selection in maintaining partial isolation of lineages, causing admixing lineages to not completely homogenize.     

Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes

Background

Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic.

Methods

RT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes.

Results

Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication.

Conclusions

Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

     

Postgenomics of Neisseria meningitidis for vaccines development

Meningococcal disease is a global problem. Multivalent (A, C, Y, W135) conjugate vaccines have been developed and licensed; however, an effective vaccine against serogroup B has not yet become available. Outer membrane vesicle (OMV) vaccines have been used to disrupt serogroup B epidemics and outbreaks. Postgenomic technologies have been useful in aiding the discovery of new protein vaccine candidates. Moreover, proteomic technologies enable large-scale identification of membrane and surface-associated proteins, and provide suitable methods to characterize and standardize the antigen composition of OMV-based vaccines.     

Chronic Inhibition,Self-Control and Eating Behavior: Test of a ‘Resource Depletion’ Model

The current research tested the hypothesis that individuals engaged in long-term efforts to limit food intake (e.g., individuals with high eating restraint) would have reduced capacity to regulate eating when self-control resources are limited. In the current research, body mass index (BMI) was used as a proxy for eating restraint based on the assumption that individuals with high BMI would have elevated levels of chronic eating restraint. A preliminary study (Study 1) aimed to provide evidence for the assumed relationship between eating restraint and BMI. Participants (N = 72) categorized into high or normal-range BMI groups completed the eating restraint scale. Consistent with the hypothesis, results revealed significantly higher scores on the weight fluctuation and concern for dieting subscales of the restraint scale among participants in the high BMI group compared to the normal-range BMI group. The main study (Study 2) aimed to test the hypothesized interactive effect of BMI and diminished self-control resources on eating behavior. Participants (N = 83) classified as having high or normal-range BMI were randomly allocated to receive a challenging counting task that depleted self-control resources (ego-depletion condition) or a non-depleting control task (no depletion condition). Participants then engaged in a second task in which required tasting and rating tempting cookies and candies. Amount of food consumed during the taste-and-rate task constituted the behavioral dependent measure. Regression analyses revealed a significant interaction effect of these variables on amount of food eaten in the taste-and-rate task. Individuals with high BMI had reduced capacity to regulate eating under conditions of self-control resource depletion as predicted. The interactive effects of BMI and self-control resource depletion on eating behavior were independent of trait self-control. Results extend knowledge of the role of self-control in regulating eating behavior and provide support for a limited-resource model of self-control.     

Flora Palinologica Italiana,Sezione Aeropalinologica — Specie esotiche - S 236:Cedrus libani A. Rich

Riassunto Nell'ambito del programma della Flora Palinologica Italiana, Sezione Aerobiologica, viene presentata la scheda palinologica diCedrus libani A. Rich. (specie esotica). Sono puntualizzate le differenze tra i parametri morfologici del polline «fresco» e acetolizzato.

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Summary According to the program «Palynological Italian Flora, Aeropalynological Section» the palynological card ofCedrus libani A. Rich. (exotic species) relevant to «fresh» and acetolyzed pollen grains is presented. The differences between the morphological parameters of fresh and acetolyzed pollen are pointed out.

     

Loss of Plastoglobule Kinases ABC1K1 and ABC1K3 Causes Conditional Degreening,Modified Prenyl-Lipids,and Recruitment of the Jasmonic Acid Pathway

Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen–derived carotenoid β-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyl-lipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wild-type PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.