Insulin-Receptor Substrate-2 (IRS-2) Is Required for Maintaining Glucokinase and Glucokinase Regulatory Protein Expression in Mouse Liver

Insulin receptor substrate (IRS) proteins play important roles in hepatic nutrient homeostasis. Since glucokinase (GK) and glucokinase regulatory protein (GKRP) function as key glucose sensors, we have investigated the expression of GK and GKRP in liver of Irs-2 deficient mice and Irs2(−/−) mice where Irs2 was reintroduced specifically into pancreatic β-cells [RIP-Irs-2/IRS-2(−/−)]. We observed that liver GK activity was significantly lower (p<0.0001) in IRS-2(−/−) mice. However, in RIP-Irs-2/IRS-2(−/−) mice, GK activity was similar to the values observed in wild-type animals. GK activity in hypothalamus was not altered in IRS-2(−/−) mice. GK and GKRP mRNA levels in liver of IRS-2(−/−) were significantly lower, whereas in RIP-Irs-2/IRS-2(−/−) mice, both GK and GKRP mRNAs levels were comparable to wild-type animals. At the protein level, the liver content of GK was reduced in IRS-2(−/−) mice as compared with controls, although GKRP levels were similar between these experimental models. Both GK and GKRP levels were lower in RIP-Irs-2/IRS-2(−/−) mice. These results suggest that IRS-2 signalling is important for maintaining the activity of liver GK. Moreover, the differences between liver and brain GK may be explained by the fact that expression of hepatic, but not brain, GK is controlled by insulin. GK activity was restored by the β-cell compensation in the RIP-Irs-2/IRS-2 mice. Interestingly, GK and GKRP protein expression remained low in RIP-Irs-2/IRS-2(−/−) mice, perhaps reflecting different mRNA half-lives or alterations in the process of translation and post-translational regulation.     

Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals

Background

Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy.

Methods

We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ΔRT and R5 envelopes: NL4-3/ΔRT/ΔEnv[AC10] and NL4-3/ΔRT/ΔEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-γ ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry.

Results

The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ΔRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: “full-responders” (positive response against both viral particles), “partial-responders” (positive response only against NL4-3/ΔRT virions) and “non-responders” (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ΔRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals.

Conclusions

In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay.     

Sonic Hedgehog Carried by Microparticles Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice

Microparticles are small fragments of the plasma membrane generated after cell stimulation. We recently showed that Sonic hedgehog (Shh) is present in microparticles generated from activated/apoptotic human T lymphocytes and corrects endothelial injury through nitric oxide (NO) release. This study investigates whether microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in mice. Male Swiss mice were implanted with osmotic minipumps delivering angiotensin II (0.5 mg/kg/day) or NaCl (0.9%). Systolic blood pressure and heart rate were measured daily during 21 days. After 7 day of minipump implantation, mice received i.v. injections of microparticles (10 µg/ml) or i.p. Shh receptor antagonist cyclopamine (10 mg/kg/2 days) during one week. Angiotensin II induced a significant rise in systolic blood pressure without affecting heart rate. Microparticles reversed angiotensin II-induced hypertension, and cyclopamine prevented the effects of microparticles. Microparticles completely corrected the impairment of acetylcholine- and flow-induced relaxation in vessels from angiotensin II-infused mice. The improvement of endothelial function induced by microparticles was completely prevented by cyclopamine treatment. Moreover, microparticles alone did not modify NO and O₂ ^{. -} production in aorta, but significantly increased NO and reduced O₂ ^{. -} productions in aorta from angiotensin II-treated mice, and these effects were blocked by cyclopamine. Altogether, these results show that microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in aorta through a mechanism associated with Shh-induced NO production and reduction of oxidative stress. These microparticles may represent a new therapeutic approach in cardiovascular diseases associated with decreased NO production.     

Shared Midgut Binding Sites for Cry1A.105, Cry1Aa,Cry1Ab,Cry1Ac and Cry1Fa Proteins from Bacillus thuringiensis in Two Important Corn Pests,Ostrinia nubilalis and Spodoptera frugiperda

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with ¹²⁵I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.     

Multi-Host Expression System for Recombinant Production of Challenging Proteins

Recombinant production of complex eukaryotic proteins for structural analyses typically requires a profound screening process to identify suitable constructs for the expression of ample amounts of properly folded protein. Furthermore, the evaluation of an optimal expression host has a major impact on protein yield and quality as well as on actual cost of the production process. Here we present a novel fast expression system for multiple hosts based on a single donor vector termed pFlp-Bac-to-Mam. The range of applications of pFlp-Bac-to-Mam comprises highly efficient transient transfection of HEK293-6E in serum-free suspension culture and subsequent large-scale production of challenging proteins expressing in mg per Liter level using either the baculoviral expression vector system or stable CHO production cell lines generated by Flp-mediated cassette exchange. The success of the multi-host expression vector to identify the optimal expression strategy for efficient production of high quality protein is demonstrated in a comparative expression study of three model proteins representing different protein classes: intracellular expression using a fluorescent protein, secretion of a single-chain-Fv-hIgG1Fc fusion construct and production of a large amount of highly homogeneous protein sample of the extracellular domain of a Toll-like receptor. The evaluation of the production efficiency shows that the pFlp-Bac-to-Mam system allows a fast and individual optimization of the expression strategy for each protein class.     

Ultrastructure of Calcareous Dinophytes (Thoracosphaeraceae,Peridiniales) with a Focus on Vacuolar Crystal-Like Particles

Biomineralization in calcareous dinophytes (Thoracosphaeracaea, Peridiniales) takes place in coccoid cells and is presently poorly understood. Vacuolar crystal-like particles as well as collection sites within the prospective calcareous shell may play a crucial role during this process at the ultrastructural level. Using transmission electron microscopy, we investigated the ultrastructure of coccoid cells at an early developmental stage in fourteen calcareous dinophyte strains (corresponding to at least ten species of Calciodinellum, Calcigonellum, Leonella, Pernambugia, Scrippsiella, and Thoracosphaera). The shell of the coccoid cells consisted either of one (Leonella, Thoracosphaera) or two matrices (Scrippsiella and its relatives) of unknown element composition, whereas calcite is deposited in the only or the outer layer, respectively. We observed crystal-like particles in cytoplasmic vacuoles in cells of nine of the strains investigated and assume that they are widespread among calcareous dinophytes. However, similar structures are also found outside the Thoracosphaeraceae, and we postulate an evolutionarily old physiological pathway (possibly involved in detoxification) that later was specialized for calcification. We aim to contribute to a deeper knowledge of the biomineralization process in calcareous dinophytes.     

Cytological Studies of Human Meiosis: Sex-Specific Differences in Recombination Originate at,or Prior to,Establishment of Double-Strand Breaks

Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB) formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC) length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.     

The res (restored cell structure by salinity) tomato mutant reveals the role of the DEAD-box RNA helicase SlDEAD39 in plant development and salt response

Increasing evidences highlight the importance of DEAD-box RNA helicases in plant development and stress responses. In a previous study, we characterized the tomato res mutant (restored cell structure by salinity), showing chlorosis and development alterations that reverted under salt-stress conditions. Map-based cloning demonstrates that RES gene encodes SlDEAD39, a chloroplast-targeted DEAD-box RNA helicase. Constitutive expression of SlDEAD39 complements the res mutation, while the silencing lines had a similar phenotype than res mutant, which is also reverted under salinity. Functional analysis of res mutant proved SlDEAD39 is involved in the in vivo processing of the chloroplast, 23S rRNA, at the hidden break-B site, a feature also supported by in vitro binding experiments of the protein. In addition, our results show that other genes coding for chloroplast-targeted DEAD-box proteins are induced by salt-stress, which might explain the rescue of the res mutant phenotype. Interestingly, salinity restored the phenotype of res adult plants by increasing their sugar content and fruit yield. Together, these results propose an unprecedented role of a DEAD-box RNA helicase in regulating plant development and stress response through the proper ribosome and chloroplast functioning, which, in turn, represents a potential target to improve salt tolerance in tomato crops.     

Prognostic factors and predictors of outcome in patients with COVID-19 and related pneumonia: a retrospective cohort study

The aim of the present study was to simultaneously assess several potential predictors of outcome (co-morbidity, previous and in-hospital treatment, radiologic Brixia score) in patients with COVID-19.This retrospective cohort study included 258 consecutive patients with confirmed COVID-19 admitted to a medical ward at Montichiari Hospital, Brescia, Italy from February 28th to April 30rd, 2020. Patients had SARS-CoV-2 related pneumonia with respiratory failure, and were treated with hydroxychloroquine and lopinavir plus ritonavir. In some patients, additional treatment with tocilizumab, dexamethasone and enoxaparin was adopted. Outcomes (death or recovery) were assessed at the end of the discharge period or at the end of the follow-up (August 2020).During hospitalization, 59 patients died, while 6 died after discharge. The following variables were demonstrated to be associated with a worse prognosis: Radiologic Brixia score higher than 8, presence at baseline of hypertension, diabetes, chronic obstructive pulmonary disease, heart disease, cancer, previous treatment with ACE-inhibitors or anti-platelet drugs. Anticoagulant treatment during hospital admission with enoxaparin at a dose higher than 4000 U once daily was associated with a better prognosis.In conclusion, our study demonstrates that some co-morbidities and cardiovascular risk factors may affect prognosis. The radiologic Brixia score may be a useful tool to stratify the risk of death at baseline. Anticoagulant treatment with enoxaparin might be associated to a clinical benefit in terms of survival in patients with COVID-19.