Simultaneous determination of carbonyl compounds related to thermal treatment and oxidative stability of infant formulas by gas-diffusion microextraction and high-performance liquid chromatography with ultraviolet detection.

Infant formulae are the only possible alternative to breastfeeding during the first year of life, so it is crucial to assure their innocuousness. Infant formula undergoes heat treatments to ensure safety and shelf life. However, such processes impact health as they lead to the formation of malondialdehyde, acrolein, and α-dicarbonyl compounds, related to Maillard reaction. Thus, there is a need for improved analytical methods to ensure the safety, quality, and nutritional value of infant formulae, and also exploring the potential of specific compounds as indicators for quality control and monitoring purposes. We developed and validated a novel, efficient, and cost-effective method using gas-diffusion microextraction for the simultaneous quantification of carbonyl compounds in infant formula. Malondialdehyde, acrolein, glyoxal, methylglyoxal, and diacetyl were detected as o-phenylenediamine derivatives using HPLC with UV detection. Parameters influencing extraction efficiency were studied using an asymmetric screening design. The validated method has shown excellent linearity, sensitivity, accuracy, and precision. It was applied to analyze 26 infant formula samples, including starter, follow-up, and special formulated powdered infant formula. Methylglyoxal was found in all samples (0.201-3.153 µg mL-1), while malondialdehyde was present only in certain starter formulas (1.033-1.802 µg mL-1). Acrolein (0.510-3.246 µg mL-1), glyoxal (0.109-1.253 µg mL-1), and diacetyl (0.119-2.001 µg mL-1) were detected in various sample types. Principal components and hierarchical cluster analyses have showcased distinct sample clustering based on analyte contents. This study presents a novel methodology for the analysis of markers of thermal treatment and oxidative stability in infant formula. It contributes to the characterization of the products' composition and quality control of infant formulae, thereby enhancing their safety and nutritional adequacy. This study also presents the first reported quantification of acrolein in infant formula and introduces the application of the acrolein-o-phenylenediamine derivative for food analysis.

Experiences and reflections about behavioral pain assays in laboratory animals.

Pharmacological assays based on the measurement of nociceptive responses in laboratory animals are a fundamental tool to assess analgesic strategies. During our experience with this type of experiments, we have been repeatedly challenged by different concerns related to their interpretation or relevance. Although these subjects are frequently discussed in our lab, they do not usually find a place in research articles with original data, in which the focus on results seems mandatory. In the present manuscript we try to discuss as central issues some of these aspects that often cross transversally our research. We have gathered them in five topics inspired by the results obtained in our laboratory. The two initial sections are devoted to the influence of the behavioral method used to assess nociception on the results achieved, as well as to the possibility that data may be more easily accepted when obtained with standard methods than with alternative ones. The third topic is related to the difficulties encountered when working with a molecule that may evoke dual effects, acting as pronociceptive or antinociceptive depending on the dose. The fourth point deals with the situation in which a particular hyperalgesic reaction is related to several molecules but the single inhibition of only one of them can completely prevent it. Finally, the last issue is addressed to comment the impact in the progress of pain research of experiments performed in animal models of pathological settings.

Involvement of CD4+ and CD8+ T-lymphocytes in the modulation of nociceptive processing evoked by CCL4 in mice.

AIMS: To explore the mechanisms involved in the transformation of analgesia produced by low doses of CCL4 (pg/kg) to hyperalgesia when higher doses (ng/kg) are administered to mice. MAIN METHODS: The unilateral hot plate test was used to assess thermal nociception. CD3+, CD4+ or CD8+ blood cells were depleted with selective antibodies. Expression of CCR5 and IL-16 in lymphocytes was studied by flow cytometry and IL-16 blood levels were measured by ELISA. IL-16 and CD8 were detected by immunofluorescence. KEY FINDINGS: IL-16 and CCR5 expression were demonstrated in CD4+ and CD8+ T-lymphocytes by flow cytometry. Furthermore, CCL4-induced hyperalgesia was abolished by reducing circulating T-lymphocyte levels or by selectively depleting CD4+ lymphocytes. In contrast, when the anti-CD4 antibody was acutely administered, CCL4 induced analgesia instead of hyperalgesia. A similar response was obtained when administering A-770041, that prevents CD4-mediated CCR5 desensitization by inhibiting p56lck kinase. As occurred with the analgesic effect evoked by low doses of CCL4, analgesia evoked by combining CCL4 and A-770041 was reverted by naloxone, naltrindole or an anti-met-enk antibody. Interestingly, flow cytometry assays showed that the number of CD8+, but not CD4+, T-cells expressing IL-16 is reduced after the acute administration of CCL4, a result compatible with the description that CD8+-lymphocytes can rapidly release preformed IL-16. Accordingly, the rise in IL-16 blood concentration evoked by CCL4 was prevented after CD8+ lymphocyte depletion. SIGNIFICANCE: CCL4-evoked hyperalgesia is related to the desensitization of CCR5 in CD4+ T-cells and to the release of IL-16 from CD8+ lymphocytes.

Chemometrics and elemental mapping by portable LIBS to identify the impact of volcanogenic and non-volcanogenic degradation sources on the mural paintings of Pompeii.

Crystallization of soluble salts is a common degradation phenomenon that threatens the mural paintings of Pompeii. There are many elements that contribute to the crystallization of salts on the walls of this archaeological site. Notably, the leachates of the pyroclastic materials ejected in 79 AD by Mount Vesuvius and local groundwater, rich in ions from the erosion of volcanic rocks. Both sources could contribute to increase the concentration of halides (fluorides and chlorides) and other salts in these walls. The distribution of volcanogenic salts and their impact on the conservation of Pompeian mural paintings have however not yet been fully disclosed. In this work, an analytical methodology useful to determine the impact of the main sources of degradation affecting the mural paintings of Pompeii is presented. This methodology combines the creation of qualitative distribution maps of the halogens (CaF and CaCl) and related alkali metals (Na and K) by portable Laser Induced Breakdown Spectroscopy (LIBS) and a subsequent Principal Component Analysis of these data. Such maps, together with the in-situ identification of sulfate salts by portable Raman spectroscopy, provided information about the migration and distribution of volcanogenic halides and the influence of ions coming from additional sources (marine aerosol and modern consolidation mortars). Additionally, the thermodynamic modeling developed using the experimentally determined ionic content of Pompeian rain- and groundwater allowed to determine their specific role in the formation of soluble salts in the mural paintings of Pompeii.

Kappa-opioid receptor-mediated thermal analgesia evoked by the intrathecal administration of the chemokine CCL1 in mice.

BACKGROUND: The chemokine CC motif ligand 1 (CCL1) participates in immune cell recruitment and, as other chemokines, is also involved in nociceptive processing. In contrast with previous reports indicating its participation in allodynia and cold hypernociception when spinally administered, its ability to evoke heat thermal analgesia, mediated by circulating leukocytes and endocannabinoids, after systemic administration has recently been reported. OBJECTIVES: Aiming to explore the role played by CCL1 on spinal nociception, we study here the effect of its intrathecal administration on thermal nociception in mice. METHODS: Behavioral nociceptive assays, immunohistochemical experiments, white cell blood depletion procedures and qRT-PCR experiments were performed. RESULTS: The intrathecal administration of CCL1 (0.3-30 ng) produced analgesia as measured by the unilateral hot plate test. This effect peaked 1 h after injection, was prevented by the CCR8 antagonist R243 and was accompanied by a reduction of c-Fos expression in spinal neurons. Whereas blood leukocyte depletion did not modify it, analgesia was abolished by the microglial inhibitor minocycline, but not the astroglial inhibitor aminoadipate. Furthermore, antinociception remained unmodified by the coadministration of cannabinoid type 1 or 2 receptors antagonists. However, it was reversed by naloxone but not by selective blockade of mu- or delta-opioid receptors. The inhibitory effect induced by the selective kappa-opioid receptor antagonist, nor-binaltorphimine, and by an anti-dynorphin A 1-17 antibody indicates that analgesia evoked by spinal CCL1 is mediated by endogenous dynorphins acting on kappa-opioid receptors. CONCLUSIONS: Endogenous dynorphin and microglia behave as key players in heat thermal analgesia evoked by spinal CCL1 in mice.

Elucidation of the Chemical Role of the Pyroclastic Materials on the State of Conservation of Mural Paintings from Pompeii.

Pyroclastic strata have always been thought to protect the archaeological remains of the Vesuvian area (Italy), hence allowing their conservation throughout the centuries. In this work, we demonstrate that they constitute a potential threat for the conservation state of the mural paintings of Pompeii. The ions that could be leached from them and the ion-rich groundwater coming from the volcanic soil/rocks may contribute to salt crystallisation. Thermodynamic modelling not only allowed to predict which salts can precipitate from such leaching events but also assisted the identification of additional sources of sulfates and alkali metals to explain the formation of the sulfates identified in efflorescences from the mural paintings of Pompeii. For the future, fluorine, mainly related to a volcanic origin, can be proposed as a marker to monitor the extent of the impact in the mural paintings of Pompeii in situ.

A low pKa ligand inhibits cancer-associated pain in mice by activating peripheral mu-opioid receptors.

The newly designed fentanyl derivative [( ±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide] (NFEPP) was recently shown to produce analgesia selectively via peripheral mu-opioid receptors (MOR) at acidic pH in rat inflamed tissues. Here, we examined the pH-dependency of NFEPP binding to brain MOR and its effects on bone cancer-induced pain in mice. The IC50 of NFEPP to displace bound [3H]-DAMGO was significantly higher compared to fentanyl at pH 7.4, but no differences were observed at pH 5.5 or 6.5. Intravenous NFEPP (30-100 nmol/kg) or fentanyl (17-30 nmol/kg) inhibited heat hyperalgesia in mice inoculated with B16-F10 melanoma cells. The peripherally-restricted opioid receptor antagonist naloxone-methiodide reversed the effect of NFEPP (100 nmol/kg), but not of fentanyl (30 nmol/kg). The antihyperalgesic effect of NFEPP was abolished by a selective MOR- (cyprodime), but not delta- (naltrindole) or kappa- (nor-binaltorphimine) receptor antagonists. Ten-fold higher doses of NFEPP than fentanyl induced maximal antinociception in mice without tumors, which was reversed by the non-restricted antagonist naloxone, but not by naloxone-methiodide. NFEPP also reduced heat hyperalgesia produced by fibrosarcoma- (NCTC 2472) or prostate cancer-derived (RM1) cells. These data demonstrate the increased affinity of NFEPP for murine MOR at low pH, and its ability to inhibit bone cancer-induced hyperalgesia through peripheral MOR. In mice, central opioid receptors may be activated by ten-fold higher doses of NFEPP.

Dual dose-related effects evoked by CCL4 on thermal nociception after gene delivery or exogenous administration in mice.

As recently described, the administration of extremely low doses (pg/kg) of CCL4 (Macrophage inflammatory protein 1ß, MIP-1ß) can induce antinociceptive effects in mice (García-Domínguez et al., 2019b). We describe here that hydrodynamic delivery of a plasmid containing CCL4 cDNA provokes a biphasic response consisting in an initial thermal hyperalgesic reaction for 8 days followed by analgesia at days 10-12, being both responses blocked after the administration of the CCR5 antagonist DAPTA. Both the luminiscence evoked in liver after the administration of a plasmid containing CCL4 and luciferase cDNAs and the hepatic concentration of CCL4 measured by ELISA were maximal 4 days after plasmid administration and markedly diminished at day 10. A dose-effect curve including a wide dose range of exogenous CCL4 revealed thermal analgesia after the administration of 10-100 pg/kg whereas 1000 times higher doses (30-100 ng/kg) induced, instead, thermal hyperalgesia inhibited by DAPTA. This hyperalgesia was absent in mice with reduced white blood cells after cyclophosphamide treatment, thus supporting the involvement of circulating leukocytes. A multiarray bioluminescent assay revealed increased plasma levels of IL-1α, CCL2, CXCL1, CXCL13, IL-16 and TIMP-1 in mice treated with 100 ng/kg of CCL4. The hyperalgesic response evoked by CCL4 was prevented by IL-1R, CXCR2 or CCR2 antagonists or by the neutralization of CXCL13 or IL-16, but not TIMP-1, with selective antibodies. The administration of the anti-IL-16 antibody was the unique treatment able to convert hyperalgesia evoked by 100 ng/kg of CCL4 in an analgesic effect. The ability of IL-16 to evoke hypernociception was confirmed by studying the response to its exogenous administration (10-30 ng/kg). In summary, the present results demonstrate that CCL4 induces a dual modulation of nociception and describe some mechanisms involved in the hyperalgesic response evoked by this chemokine.

The Systemic Administration of the Chemokine CCL1 Evokes Thermal Analgesia in Mice Through the Activation of the Endocannabinoid System.

Apart from its involvement in immune functions, the chemokine CCL1 can participate in the modulation of nociceptive processing. Previous studies have demonstrated the hypernociceptive effect produced by CCL1 in the spinal cord, but its possible action on peripheral nociception has not yet been characterized. We describe here that the subcutaneous administration of CCL1 (1-10 µg/kg) produces dose-dependent and long-lasting increases in thermal withdrawal latencies measured by the unilateral hot plate test in mice. The antinociceptive nature of this effect is further supported by the reduction of spinal neurons expressing Fos protein in response to a noxious thermal stimulus observed after the administration of 10 µg/kg of CCL1. CCL1-induced antinociception was inhibited after systemic, but not spinal administration of the selective antagonist R243 (0.1-1 mg/kg), demonstrating the participation of peripheral CCR8 receptors. The absence of this analgesic effect in mice treated with a dose of cyclophosphamide that produces a drastic depletion of leukocytes suggests its dependency on white blood cells. Furthermore, whereas the antinociceptive effect of CCL1 was unaffected after the treatment with either the antagonist of opioid receptors naloxone or the cannabinoid type 1 receptor blocker AM251, it was dose-dependently inhibited after the administration of the CB2 receptor antagonist SR144528 (0.1-1 mg/kg). The detection by ELISA of an increased presence of the endocannabinoid 2-arachidonoylglycerol after the administration of an analgesic dose of CCL1 supports the notion that CCL1 can evoke thermal analgesia through the release of this endocannabinoid from circulating leukocytes.

Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

Robustness of signal detection in cryo-electron microscopy via a bi-objective-function approach.

BACKGROUND: The detection of weak signals and selection of single particles from low-contrast micrographs of frozen hydrated biomolecules by cryo-electron microscopy (cryo-EM) represents a major practical bottleneck in cryo-EM data analysis. Template-based particle picking by an objective function using fast local correlation (FLC) allows computational extraction of a large number of candidate particles from micrographs. Another independent objective function based on maximum likelihood estimates (MLE) can be used to align the images and verify the presence of a signal in the selected particles. Despite the widespread applications of the two objective functions, an optimal combination of their utilities has not been exploited. Here we propose a bi-objective function (BOF) approach that combines both FLC and MLE and explore the potential advantages and limitations of BOF in signal detection from cryo-EM data. RESULTS: The robustness of the BOF strategy in particle selection and verification was systematically examined with both simulated and experimental cryo-EM data. We investigated how the performance of the BOF approach is quantitatively affected by the signal-to-noise ratio (SNR) of cryo-EM data and by the choice of initialization for FLC and MLE. We quantitatively pinpointed the critical SNR (~ 0.005), at which the BOF approach starts losing its ability to select and verify particles reliably. We found that the use of a Gaussian model to initialize the MLE suppresses the adverse effects of reference dependency in the FLC function used for template-matching. CONCLUSION: The BOF approach, which combines two distinct objective functions, provides a sensitive way to verify particles for downstream cryo-EM structure analysis. Importantly, reference dependency of the FLC does not necessarily transfer to the MLE, enabling the robust detection of weak signals. Our insights into the numerical behavior of the BOF approach can be used to improve automation efficiency in the cryo-EM data processing pipeline for high-resolution structural determination.

The credibility of scientific communication sources regarding climate change: A population-based survey experiment.

This article analyses whether different institutional sources of scientific information have an impact on its credibility. Through a population-based survey experiment of a national representative sample of the Spanish public, we measure the credibility that citizens attribute to scientific information on the evolution of CO2 emissions disclosed by different institutional sources (business associations, government, non-government environmental organisations, international bodies and national research institutions). The findings show that an institutional credibility gap exists in science communication. We also investigate the factors accounting for the credibility of the different institutional sources by examining variables related to knowledge, interest, trust, reputation, deference, attitudes, values and personal characteristics. Exploratory regression analyses reveal that identical variables can produce different effects on the credibility of scientific information, depending on the institutional source to which it is attributed.

The Chemokine CCL4 (MIP-1ß) Evokes Antinociceptive Effects in Mice: a Role for CD4+ Lymphocytes and Met-Enkephalin.

In the present study, we characterize the antinociceptive effects produced by the chemokine CCL4 in mice. The intraplantar administration of very low doses of CCL4 (0.1-3 pg) produced bilateral antinociception assessed by the unilateral hot-plate test (UHP) without evoking chemotactic responses at the injection site. Moreover, the subcutaneous administration of CCL4 (3-100 pg/kg) also yielded bilateral antinociception in the UHP and the paw pressure test and reduced the number of spinal neurons that express Fos protein in response to noxious stimulation. The implication of peripheral CCR5 but not CCR1 in CCL4-evoked antinociception was deduced from the inhibition produced by systemic but not intrathecal, administration of the CCR5 antagonist DAPTA, and the inefficacy of the CCR1 antagonist J113863. Besides, the inhibition observed after subcutaneous but not intrathecal administration of naloxone demonstrated the involvement of peripheral opioids and the efficacy of naltrindole but not cyprodime or nor-binaltorphimine supported the participation of δ-opioid receptors. In accordance, plasma levels of met-enkephalin, but not ß-endorphin, were augmented in response to CCL4. Likewise, CCL4-evoked antinociception was blocked by the administration of an anti-met-enk antibody. Leukocyte depletion experiments performed with cyclophosphamide, anti-Ly6G, or anti-CD3 antibodies indicated that the antinociceptive effect evoked by CCL4 depends on circulating T lymphocytes. Double immunofluorescence experiments showed a four times more frequent expression of met-enk in CD4+ than in CD8+ T lymphocytes. CCL4-induced antinociception almost disappeared upon CD4+, but not CD8+, lymphocyte depletion with selective antibodies, thus supporting that the release of met-enk from CD4+ lymphocytes underlies the opioid antinociceptive response evoked by CCL4.

Conformational Differences between Functional Human Immunodeficiency Virus Envelope Glycoprotein Trimers and Stabilized Soluble Trimers.

Binding to the receptor CD4 triggers entry-related conformational changes in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, (gp120/gp41)3 Soluble versions of HIV-1 Env trimers (sgp140 SOSIP.664) stabilized by a gp120-gp41 disulfide bond and a change (I559P) in gp41 have been structurally characterized. Here, we use cross-linking/mass spectrometry to evaluate the conformations of functional membrane Env and sgp140 SOSIP.664. Differences were detected in the gp120 trimer association domain and C terminus and in the gp41 heptad repeat 1 (HR1) region. Whereas the membrane Env trimer exposes the gp41 HR1 coiled coil only after CD4 binding, the sgp140 SOSIP.664 HR1 coiled coil was accessible to the gp41 HR2 peptide even in the absence of CD4. Our results delineate differences in both gp120 and gp41 subunits between functional membrane Env and the sgp140 SOSIP.664 trimer and provide distance constraints that can assist validation of candidate structural models of the native HIV-1 Env trimer.IMPORTANCE HIV-1 envelope glycoprotein spikes mediate the entry of the virus into host cells and are a major target for vaccine-induced antibodies. Soluble forms of the envelope glycoproteins that are stable and easily produced have been characterized extensively and are being considered as vaccines. Here, we present evidence that these stabilized soluble envelope glycoproteins differ in multiple respects from the natural HIV-1 envelope glycoproteins. By pinpointing these differences, our results can guide the improvement of envelope glycoprotein preparations to achieve greater similarity to the viral envelope glycoprotein spike, potentially increasing their effectiveness as a vaccine.

SOSIP Changes Affect Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Conformation and CD4 Engagement.

The entry of human immunodeficiency virus into host cells is mediated by the envelope glycoprotein (Env) trimeric spike, which consists of three exterior gp120 subunits and three transmembrane gp41 subunits. The trimeric Env undergoes extensive conformational rearrangement upon interaction with the CD4 receptor, transitioning from the unliganded, "closed" State 1 to more-open downstream State 2 and State 3 conformations. Changes in "restraining" amino acid residues, such as leucine 193 and isoleucine 423, destabilize State 1 Env, which then assumes entry-competent, downstream conformations. The introduction of an artificial disulfide bond linking the gp120 and gp41 subunits (SOS) in combination with the I559P (IP) change has allowed structural characterization of soluble gp140 (sgp140) trimers. The conformation of these SOSIP-stabilized sgp140 trimers has been suggested to represent the closed native State 1 conformation. Here we compare the impact on the membrane Env conformation of the SOSIP changes with that of the well-characterized changes (L193R and I423A) that shift Env to downstream States 2 and 3. The results presented here suggest that the SOSIP changes stabilize Env in a conformation that differs from State 1 but also from the downstream Env conformations stabilized by L193R or I423A.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is triggered by receptor binding to mediate the entry of the virus into cells. Most structural studies of Env trimers have utilized truncated soluble gp140 Envs stabilized with the I559P and SOS changes. Here we present evidence indicating that these stabilizing changes have a profound impact on the conformation of Env, moving Env away from the native pretriggered Env conformation. Our studies underscore the need to acquire structural information on the pretriggered Env conformation, which is recognized by most broadly reactive neutralizing antibodies.

Autonomy and Authority in Public Research Organisations: Structure and Funding Factors.

This paper establishes a structural typology of the organisational configurations of public research organisations which vary in their relative internal sharing of authority between researchers and managers; we distinguish between autonomous, heteronomous and managed research organisations. We assume that there are at least two sources of legitimate authority within research organisations, one derived from formal hierarchy (organisational leadership) and another derived from the research community (professional); the balance of authority between researchers and managers is essentially structural but is empirically mediated by the funding portfolio of organisations and the corresponding endowment of resources at the disposal of leaders or researchers. Changes in the level, sources and strings of organisational and individual research funding are expected to affect the balance of internal authority in different ways depending on the organisational configuration, and to open the door to the influence of external actors in the development of research agendas.

Comparison of Uncleaved and Mature Human Immunodeficiency Virus Membrane Envelope Glycoprotein Trimers.

The mature envelope glycoprotein (Env) spike on the surfaces of human immunodeficiency virus type 1 (HIV-1)-infected cells and virions is derived from proteolytic cleavage of a trimeric gp160 glycoprotein precursor. In these studies, we compared the conformations of cleaved and uncleaved membrane Envs with truncated cytoplasmic tails to those of stabilized soluble gp140 SOSIP.664 Env trimers. Deletion of the gp41 cytoplasmic tail did not significantly affect the sensitivity of viruses with the HIV-1AD8 Env to inhibition by antibodies or a CD4-mimetic compound. After glutaraldehyde fixation and purification from membranes, a cleaved Env exhibited a hydrodynamic radius of ∼10 nm and an antibody-binding profile largely consistent with that expected based on virus neutralization sensitivity. The purified cleaved Env trimers exhibited a hollow architecture with a central void near the trimer axis. Uncleaved Env, cross-linked and purified in parallel, exhibited a hydrodynamic radius similar to that of the cleaved Env. However, the uncleaved Env was recognized by poorly neutralizing antibodies and appeared by negative-stain electron microscopy to sample multiple conformations. Compared with membrane Envs, stabilized soluble gp140 SOSIP.664 Env trimers appear to be more compact, as reflected in their smaller hydrodynamic radii and negative-stain electron microscopy structures. The antigenic features of the soluble gp140 SOSIP.664 Env trimers differed from those of the cleaved membrane Env, particularly in gp120 V3 and some CD4-binding-site epitopes. Thus, proteolytic maturation allows the membrane-anchored Env to achieve a conformation that retains functional metastability but masks epitopes for poorly neutralizing antibodies.IMPORTANCE The entry of human immunodeficiency virus type 1 (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) spike on the surface of the virus. Host antibodies elicited during natural HIV-1 infection or by vaccination can potentially recognize the Env spike and block HIV-1 infection. However, the changing shape of the HIV-1 Env spike protects the virus from antibody binding. Understanding the shapes of natural and man-made preparations of HIV-1 Envs will assist the development of effective vaccines against the virus. Here, we evaluate the effects of several Env modifications commonly used to produce Env preparations for vaccine studies and the determination of structure. We found that the cleavage of the HIV-1 Env precursor helps Env to assume its natural shape, which resists the binding of many commonly elicited antibodies. Stabilized soluble Envs exhibit more compact shapes but expose some Env elements differently than the natural Env.

Synergistic combinations of the dual enkephalinase inhibitor PL265 given orally with various analgesic compounds acting on different targets, in a murine model of cancer-induced bone pain.

BACKGROUND: The first line pharmacological treatment of cancer pain is morphine and surrogates but a significant pain relief and a reduction of the side-effects of these compounds makes it necessary to combine them with other drugs acting on different targets. The aim of this study was to measure the antinociceptive effect on cancer-induced bone pain resulting from the association of the endogenous opioids enkephalin and non-opioid analgesic drugs. For this purpose, PL265 a new orally active single dual inhibitor of the two degrading enkephalins enzymes, neprilysin (NEP) and aminopeptidase N (APN) was used. It strictly increased the levels of enkephalin at their sites of releases. The selected non-opioid compounds are: gabapentin, A-317491 (P2X3 receptor antagonist), ACEA (CB1 receptor antagonist), AM1241 (CB2 receptor antagonist), JWH-133 (CB2 receptor antagonist), URB937 (FAAH inhibitor), and NAV26 (Nav1.7 channel blocker). METHODS: Experiments. Experiments were performed in 5-6 weeks old (26-33g weight) C57BL/6 mice. Cell culture and cell inoculation. B16-F10 melanoma cells were cultured and when preconfluent, treated and detached. Finally related cells were resuspended to obtain a concentration of 2×106 cells/100µL. Then 105 cells were injected into the right tibial medullar cavity. Control mice were treated by killed cells by freezing. Behavioural studies. Thermal withdrawal latencies were measured on a unilatered hot plate (UHP) maintained at 49±0.2°C. Mechanical threshold values were obtained by performing the von Frey test using the "up and down" method. To evaluate the nature (additive or synergistic) of the interactions between PL265 and different drugs, an isobolographic analysis following the method described by Tallarida was performed. RESULTS: The results demonstrate the ability of PL265, a DENKI that prevents the degradation of endogenous ENKs, to counteract cancer-induced bone thermal hyperalgesia in mice, by exclusively stimulating peripheral opioid receptors as demonstrated by used of an opioid antagonist unable to enter the brain. The development of such DENKIs, endowed with druggable pharmacokinetic characteristics, such as good absorption by oral route, can be considered as an important step in the development of much needed novel antihyperalgesic drugs. Furthermore, all the tested combinations resulted in synergistic antihyperalgesic effects. As shown here, the greatest synergistic antinociceptive effect (doses could be lowered by 70%) was produced by the combination of PL265 with the P2X3 receptor antagonist (A-317491), cannabinoid CB1 receptor agonist (exogenous, ACEA and endogenous URB937-protected-AEA) and Nav1.7 blocker (NAV26) whose mechanism of action involves the direct activation of the enkephalinergic system. CONCLUSIONS: These multi-target-based antinociceptive strategies using combinations of non-opioid drugs with dual inhibitors of enkephalin degrading enzymes may bring therapeutic advantages in terms of efficacy and safety by allowing the reduction of doses of one of the compounds or of both, which is of the utmost interest in the chronic treatment of cancer pain. IMPLICATIONS: This article presents synergistic antinociceptive effect produced by the combination of PL265 with non-opioid analgesic drugs acting via unrelated mechanisms. These multi-target-based antinociceptive strategies may bring therapeutic advantages by allowing the reduction of doses, which is of great interest in the chronic treatment of cancer pain.

Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs.

The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context.

Hyperalgesic and hypoalgesic mechanisms evoked by the acute administration of CCL5 in mice.

We show here that the intraplantar administration of CCL5 in mice produces hyperalgesia at low doses but activates compensatory antinociceptive mechanisms at doses slightly higher. Thus, the injection of 3-10ng of CCL5 evoked thermal hyperalgesia through the activation of CCR1 and CCR5 receptors, as demonstrated by the inhibitory effect exerted by the selective antagonists J113863 (0.01-0.1µg) and DAPTA (0.3-3µg), respectively. The prevention of this hyperalgesia by diclofenac (1-10µg), the inhibitors of COX-1 SC-560 (0.1-1µg) or COX-2 celecoxib (1-5µg), the TRPV1 antagonist capsazepine (0.03-0.3µg) or the TRPA1 antagonist HC030031 (10-50µg) demonstrates the involvement of prostaglandin synthesis and TRP sensitization in CCL5-evoked hyperalgesia. Doses of CCL5 higher than 17µg did not evoke hyperalgesia. However, this effect was restored by the administration of naloxone-methiodide (5µg), nor-binaltorphimine (10mg/kg) or an anti-dynorphin A antibody (0.62-2.5ng). The administration of 30ng of CCL5 also induced hyperalgesia in mice with reduced number of circulating white blood cells in response to cyclophosphamide or with selective neutrophil depletion induced by an anti-Ly6G antibody. In fact, the number of neutrophils present in paws treated with 30ng of CCL5 was greater than in paws receiving the administration of the hyperalgesic dose of 10ng. Finally, the expression of the endogenous opioid peptide dynorphin A was demonstrated by double immunofluorescence assays in these neutrophils attracted by CCL5. These results support previous data describing the hyperalgesic properties of CCL5 and constitute the first indication that a chemokine of the CC group can activate endogenous analgesic mechanisms.