Cell-free assays reveal the HIV-1 capsid protects reverse transcripts from cGAS.

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect their genomes from cGAS even after completion of reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, capsid inhibitors also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

A Two-Year Retrospective Analysis of the Clinical Outcomes of Immediate Submuscular Breast Reconstructions With Native® Acellular Dermal Matrix.

BACKGROUND: Implant-based submuscular breast reconstruction (SBR) can be performed with the aid of acellular dermal matrices (ADM) for implant coverage on their inferolateral pole, aiming at providing a biological interface for hiding the implant and therefore reducing the risk of complications. The purpose of this study is to assess the long-term post-operative outcomes obtained using the SBR-specific Native® ADM (DECO med s.r.l., Marcon, Venice, Italy). METHODS: All cases of Native®-assisted immediate SBR performed at our institution between October 2016 and March 2020 were retrospectively analysed. Demographic and surgical data were collected, and post-operative outcomes, including minor and major complications, were evaluated. Particular attention was paid to complications emerging before and after patient discharge. Dependence analyses were performed to uncover statistically significant relationships between risk factors and reconstructive outcomes. RESULTS: Data on 100 patients were collected, for a total of 128 breasts. The mean age of the cohort was 49.5 years, the mean BMI was 23.4 kg/m2, and the mean follow-up was 24 months. Out of this, 14.1% of patients received pre-operative radiotherapy, while 16.4% underwent post-mastectomy radiotherapy. Breasts appeared to develop short-term minor complications more likely during hospitalisation (11.7% vs. 7.8%), while short-term major complications occurred more often after discharge (7.8% vs. 15.6%). The most frequent long-term complications were capsular contracture and contour defects (both 9.4%). Risk factors that showed a statistically significant relationship with complications were pre- and post-mastectomy radiotherapy and post-operative chemotherapy. CONCLUSIONS: The retrospective analysis showed results in line with clinical outcomes reported in the literature for the same reconstructive technique. The use of Native® ADM in SBR is safe and effective in the long term.

Validation of the Portuguese self-administered computerised 24-hour dietary recall among second-, third- and fourth-grade children.

BACKGROUND: Current methods for assessing children's dietary intake, such as interviewer-administered 24-h dietary recall (24-h DR), are time consuming and resource intensive. Self-administered instruments offer a low-cost diet assessment method for use with children. The present study assessed the validity of the Portuguese self-administered, computerised, 24-h DR (PAC24) against the observation of school lunch. METHODS: Forty-one, 7-10-year-old children from two elementary schools, in Lisbon, were observed during school lunch followed by completion of the PAC24 the next day. Accuracy for reporting items was measured in terms of matches, intrusions and omissions; accuracy for reporting amounts was measured in terms of arithmetic and absolute differences for matches and amounts for omissions and intrusions; and accuracy for reporting items and amounts combined was measured in terms of total inaccuracy. The ratio of the estimated weight of food consumed with the actual weight consumed was calculated along with the limits of agreement using the method of Bland and Altman. RESULTS: Comparison of PAC24 against observations at the food level resulted in values of 67.0% for matches, 11.5% for intrusions and 21.5% for omissions. The mean for total inaccuracy was 3.44 servings. For amounts, accuracy was high for matches (-0.17 and 0.23 servings for arithmetic and absolute differences, respectively) and lower for omissions (0.61 servings) and intrusions (0.55 servings). PAC24 was found to under-estimate the weight of food on average by 32% of actual intake. CONCLUSIONS: PAC24 is a lower-burden procedure for both respondents and researchers and, with slight modification, comprises a promising method for assessing diet among children.

Nondenaturing purification of co-transcriptionally folded RNA avoids common folding heterogeneity.

Due to the energetic frustration of RNA folding, tertiary structured RNA is typically characterized by a rugged folding free energy landscape where deep kinetic barriers separate numerous misfolded states from one or more native states. While most in vitro studies of RNA rely on (re)folding chemically and/or enzymatically synthesized RNA in its entirety, which frequently leads into kinetic traps, nature reduces the complexity of the RNA folding problem by segmental, co-transcriptional folding starting from the 5' end. We here have developed a simplified, general, nondenaturing purification protocol for RNA to ask whether avoiding denaturation of a co-transcriptionally folded RNA can reduce commonly observed in vitro folding heterogeneity. Our protocol bypasses the need for large-scale auxiliary protein purification and expensive chromatographic equipment and involves rapid affinity capture with magnetic beads and removal of chemical heterogeneity by cleavage of the target RNA from the beads using the ligand-induced glmS ribozyme. For two disparate model systems, the Varkud satellite (VS) and hepatitis delta virus (HDV) ribozymes, we achieve >95% conformational purity within one hour of enzymatic transcription, without the need for any folding chaperones. We further demonstrate that in vitro refolding introduces severe conformational heterogeneity into the natively-purified VS ribozyme but not into the compact, double-nested pseudoknot fold of the HDV ribozyme. We conclude that conformational heterogeneity in complex RNAs can be avoided by co-transcriptional folding followed by nondenaturing purification, providing rapid access to chemically and conformationally pure RNA for biologically relevant biochemical and biophysical studies.

Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis.

Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.

Biomonitoring of PCDD/Fs in populations living near portuguese solid waste incinerators: levels in human milk.

In the context of two Environmental Health Surveillance Programs, launched in response to public and scientific concern in relation to waste incinerators located near Lisbon and in Madeira Island, two human biomonitoring projects have been started in Portugal, focussed in dioxins and dioxin-like compounds in human milk. Results from the undertaken studies have already provided data on the extent and pattern of dioxin body burden of both studied groups as well as a preliminary temporal trend of dioxin levels for the population residing near Lisbon incinerator. The present paper investigates difference between exposed and non-exposed subjects under study and, from a preventive perspective, possible covariates of the dioxin levels in human milk. Emissions from both incinerators appear to be well controlled as there is no increase of human body burden of dioxins as measured in human milk of individuals living near these facilities. Concerning other determinants of dioxin levels, results suggest confirmation of previously found significant age-dependent trend towards higher levels of dioxins in aged subjects. On the contrary, association between mother's levels of dioxins and parity lost significance. Apart from the issue of incineration, the general conclusion for the general population is that living in Lisbon as compared to Madeira results in higher milk dioxin levels and possible health risks. The profile of the single congeners for PCDD/Fs in human milk from Madeira and Lisbon shows similar contributions for 12378-PCDD, 23478-PCDF, 123678-HCDD and 2378-TCDD, that account altogether for about 84% of the total identified dioxin body burden in the studied groups.

Solution probing of metal ion binding by helix 27 from Escherichia coli 16S rRNA.

Helix (H)27 from Escherichia coli 16S ribosomal (r)RNA is centrally located within the small (30S) ribosomal subunit, immediately adjacent to the decoding center. Bacterial 30S subunit crystal structures depicting Mg(2+) binding sites resolve two magnesium ions within the vicinity of H27: one in the major groove of the G886-U911 wobble pair, and one within the GCAA tetraloop. Binding of such metal cations is generally thought to be crucial for RNA folding and function. To ask how metal ion-RNA interactions in crystals compare with those in solution, we have characterized, using solution NMR spectroscopy, Tb(3+) footprinting and time-resolved fluorescence resonance energy transfer (tr-FRET), location, and modes of metal ion binding in an isolated H27. NMR and Tb(3+) footprinting data indicate that solution secondary structure and Mg(2+) binding are generally consistent with the ribosomal crystal structures. However, our analyses also suggest that H27 is dynamic in solution and that metal ions localize within the narrow major groove formed by the juxtaposition of the loop E motif with the tandem G894-U905 and G895-U904 wobble pairs. In addition, tr-FRET studies provide evidence that Mg(2+) uptake by the H27 construct results in a global lengthening of the helix. We propose that only a subset of H27-metal ion interactions has been captured in the crystal structures of the 30S ribosomal subunit, and that small-scale structural dynamics afforded by solution conditions may contribute to these differences. Our studies thus highlight an example for differences between RNA-metal ion interactions observed in solution and in crystals.

Dynamics inherent in helix 27 from Escherichia coli 16S ribosomal RNA.

The original interpretation of a series of genetic studies suggested that the highly conserved Escherichia coli 16S ribosomal RNA helix 27 (H27) adopts two alternative secondary structure motifs, the 885 and 888 conformations, during each cycle of amino acid incorporation. Recent crystallographic and genetic evidence has called this hypothesis into question. To ask whether a slippery sequence such as that of H27 may harbor inherent conformational dynamics, we have designed a series of model RNAs based on E. coli H27 for in vitro physicochemical studies. One-dimensional (1)H NMR spectroscopy demonstrates that both the 885 and 888 conformations are occupied to approximately the same extent (f(888) = 0.427 +/- 0.04) in the native H27 sequence at low pH (6.4) and low ionic strength (50 mM NaCl). UV irradiation assays conducted under conditions analogous to those used for assays of ribosomal function (pH 7.5 and 20 mM MgCl(2)) suggest that nucleotides 892 and 905, which are too far apart in the known 885 crystal structures, can approach each other closely enough to form an efficient cross-link. The use of a fluorescence resonance energy transfer (FRET)-labeled RNA together with a partially complementary DNA oligonucleotide that induces a shift to the 888 conformation shows that H27 interchanges between the 885 and 888 conformations on the millisecond time scale, with an equilibrium constant of 0.33 +/-0.12. FRET assays also show that tetracycline interferes with the induced shift to the 888 conformation, a finding that is consistent with crystallographic localization of tetracycline bound to the 885 conformation of H27 in the 30S ribosomal subunit. Taken together, our data demonstrate the innate tendency of an isolated H27 to exist in a dynamic equilibrium between the 885 and 888 conformations. This begs the question of how these inherent structural dynamics are suppressed within the context of the ribosome.

Correlating structural dynamics and function in single ribozyme molecules.

We have studied the correlation between structural dynamics and function of the hairpin ribozyme. The enzyme-substrate complex exists in either docked (active) or undocked (inactive) conformations. Using single-molecule fluorescence methods, we found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also found substrate cleavage to be rate-limited by a combination of conformational transitions and reversible chemistry equilibrium. The complex structural dynamics quantitatively explain the heterogeneous cleavage kinetics common to many catalytic RNAs. The intimate coupling of structural dynamics and function is likely a general phenomenon for RNA.

Reaction pathway of the trans-acting hepatitis delta virus ribozyme: a conformational change accompanies catalysis.

The hepatitis delta virus (HDV), an infectious human pathogen and satellite of hepatitis B virus, leads to intensified disease symptoms, including progression to liver cirrhosis. Both the circular RNA genome of HDV and its complementary antigenome contain the same cis-cleaving catalytic RNA motif that plays a crucial role in virus replication. Previously, the high-resolution crystal structure of the product form of a cis-acting genomic HDV ribozyme has been determined, while a trans-acting version of the ribozyme was used to dissect the cleavage reaction pathway. Using fluorescence resonance energy transfer (FRET) on a synthetic trans-cleaving form of the ribozyme, we are able to directly observe substrate binding (at a rate constant k(on) of 7.8 x 10(6) M(-1) min(-1) at pH 7.5, 11 mM MgCl(2), and 25 degrees C) and dissociation (at 0.34 min(-1)). Steady-state and time-resolved FRET experiments in solution and in nondenaturing gels reveal that the substrate (precursor) complex is slightly more compact (by approximately 3 A) than the free ribozyme, yet becomes significantly extended (by approximately 15 A) upon cleavage and product complex formation. We also find that trans cleavage is characterized by a high transition-state entropy (-26 eu). We propose that the significant global conformational change that we observe between the precursor and product structures occurs on the reaction trajectory into a constrained product complex-like transition state. Our observations may present the structural basis of the recently described utilization of intrinsic substrate binding energy to the overall catalytic rate enhancement by the trans-acting HDV ribozyme.

Isradipine in the treatment of mild-to-moderate hypertension in geriatric patients.

A number of studies have shown the benefit of hypertensive treatment even though the most common forms of the disease are mild-to-moderate in severity. Considering the overall aging of the world's population, it is of particular interest to study hypertension and its treatment in geriatric patients. A short-term study of isradipine was conducted to assess its effectiveness and tolerability in patients with mild-to-moderate hypertension. The study was carried out by general practitioners and involved 3343 patients, aged > or = 18 years, with diastolic blood pressures (DBPs) ranging from 95 to 114 mm Hg. A 4-week wash-out and placebo run-in phase was followed by a 12-week active treatment period with isradipine at 1.25 or 2.5 mg/day, depending on the blood pressure response. Posttreatment results in a subgroup of 1092 patients (444 men and 648 women), aged > or = 60 years, showed decreases in systolic blood pressure (SBP) from 173.1 to 149.2 mm Hg (mean decrease, 20.9 mm Hg) and, in DBP, from 102.0 to 85.0 mm Hg (mean decrease, 16.9 mm Hg). The majority (84.6%) of these patients showed DBP reductions of > 10 mm Hg, and 82.3% achieved normalization (DBP < 90 mm Hg) at the end of treatment. The mean dosage was 1.74 +/- 0.69 mg twice daily, and 37% of patients doubled their initial 1.25 mg twice daily dosages. There were no significant changes in either heart rate or major metabolic parameters. Adverse events were reported by 3.1% of the patients, and 90% of both patients and physicians expressed satisfaction with the therapy. There were no differences between men and women with regard to adverse events or efficacy, nor were the results in patients > or = 60 years different from those in younger patients. Thus, isradipine was effective and well tolerated in these geriatric patients.

Isradipine in the treatment of mild-to-moderate hypertension in Portugal.

A short-term trial of isradipine was conducted to assess its effectiveness and tolerability in the treatment of mild-to-moderate hypertension. The study was carried out by general practitioners and involved 2,702 patients, aged 18-70 years, who had diastolic blood pressures (DBP) of 95-114 mm Hg. Patients completed a pretreatment phase of up to 4 weeks for antihypertensive drug washout and placebo run-in, before entering a 12-week active-treatment phase with 1.25 mg of isradipine twice daily, which was increased after 4 weeks to 2.5 mg twice daily, depending on the blood pressure response. At the end of 12 weeks, the mean systolic blood pressure (SBP) and DBP were 148.1 and 86.7 mm Hg compared with 169.0 and 103.0 mm Hg after placebo, respectively. The majority of patients (89.6%) had a DBP reduction greater than 10 mm Hg, and 86.2% had normalized DBP (less than or equal to 90 mm Hg) at the end of treatment. Adverse events were reported by 2.8% of patients, and 90% of patients and general practitioners reported satisfaction with the treatment. Thus, our results indicate that isradipine is effective and well tolerated, and may deserve a place as first-line treatment in mild-to-moderate hypertension.