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IMPORTANCE: Astronauts returning from long-duration spaceflight experience ocular remodeling related to cephalad fluid shifts induced by microgravity. It is hypothesized that the absence of diurnal reductions in intracranial pressure in microgravity creates a low but persistent pressure gradient at the posterior aspect of the eye, which results in ocular remodeling and space-associated neuro-ocular syndrome (SANS) over many months. OBJECTIVE: To determine whether partial reintroduction of footward fluid shifts during simulated microgravity via lower body negative pressure (LBNP) during sleep attenuates choroid engorgement, an early marker of ocular remodeling related to SANS. DESIGN, SETTING, AND PARTICIPANTS: Between May 2019 and February 2020, participants with no major cardiovascular, kidney, or ophthalmic disease completed 3 days of supine (0°) bed rest with and 3 days without 8 hours of nightly LBNP in a randomized, crossover design. This single-center investigation took place at the UT Southwestern Medical Center. All analyses were conducted blinded to condition and time point. INTERVENTIONS: Eight hours of nightly LBNP (−20 mm Hg) vs no LBNP. MAIN OUTCOMES AND MEASURES: The primary outcome was the change in choroid area and volume after 3 days of bed rest measured by optical coherence tomography. RESULTS: Of 10 participants, 5 were female, the mean (SD) age was 29 (9) years, and the age range was 18 to 55 years. Central venous pressure increased from the seated to supine position (mean [SD], seated: −2.3 [2.0] vs supine: 6.9 [2.0] mm Hg; P

Ionizing radiation is nowadays effectively used in cancer treatments. However, the effect of irradiation in immune-system cells is poorly understood and remains controversial. The aim of this work was to determine the effect of γ-irradiation in the structural and functional properties of mice splenic cells.Structural traits of irradiated splenic cells were evaluated by Atomic Force Microscopy and Raman spectroscopy. Functional properties were measured by gene and protein expression by RT-qPCR and ELISA, respectively. The induced cytotoxic effect was evaluated by MTT assay and the phagocytic capability by flow cytometry.Membrane roughness and molecular composition of splenic adherent cells are not changed by irradiation doses exposure. An increase in transcription of pro-inflammatory cytokines was observed. While protein expression decreased in IL-2 dose-dependent, relevant differences were identified in the anti-inflammatory marker IL-10 at 27 Gy. An increase of cytotoxicity in irradiated cells at 7 Gy and 27 Gy doses was observed, while phagocytosis was slight increased at 7 Gy dose but not statistically significant.We have demonstrated that γ-irradiation affects the splenic cells and changes the cytokines profile toward a pro-inflammatory phenotype and a tendency to increase the cytotoxicity was found, which implies a stimulation of immune response induced by γ-irradiation.

Molecular fingerprints revealed by Raman techniques show great potential for biomedical applications, like disease diagnostic through Raman detection of tumor markers and other molecules in the cell membrane. However, SERS substrates used in membrane molecule studies produce enhanced Raman spectra of high variability and challenging band assignments that limit their application. In this work, these drawbacks are addressed to detect membrane-associated hemoglobin (Hbm) in human erythrocytes through Raman spectroscopy. These cells are incubated with silver nanoparticles (AgNPs) in PBS before Raman measurements. Our results showed that AgNPs form large aggregates in PBS that adhered to the erythrocyte membrane, which enhances Raman scattering by molecules around the membrane, like Hbm. Also, deoxyHb markers may allow Hbm detection in Raman spectra of oxygenated erythrocytes (oxyRBCs). Raman spectra of oxyRBCs incubated with AgNPs showed enhanced deoxyHb signals with good spectral reproducibility, supporting the Hbm detection through deoxyHb markers. Instead, Raman spectra of oxyRBCs showed oxyHb bands associated with free cytoplasmic hemoglobin. Other factors influencing Raman detection of membrane proteins are discussed, like both z-position and dimension of the sample volume. The results encourage membrane protein studies in living cells using Raman spectroscopy, leading to the characterization and diagnostic of different pathologies through a non-invasive technique.

Storage and ionizing radiation of human red blood cells (RBC) produce alterations on RBC membranes and modify their normal shape and functionality. We investigated early morphological and biochemical changes in RBC due to those stressing agents at the nanoscale level and their impact on blood quality.Whole blood samples from healthy donors were γ-irradiated with 15, 25, 35, and 50 Gy. Non-irradiated and non-stored RBC were used as control samples. Irradiated blood samples were stored separately at 4 °C and analyzed immediately and after 5 and 13 d. Atomic force microscopy (AFM), osmotic fragility and Raman spectroscopy were used to detect morphological and biochemical changes.RBC function is challenged by both irradiation and storage. The storage procedure caused nanometric variations over the surface of RBC membrane for both irradiated and non-irradiated cells. The membrane of RBC became more fragile, while the biochemical fingerprint of hemoglobin (Hb) remained unaltered.Our work shows that the irradiation procedure leads to an increase in the number and size of nanovesicles along with the dose. The functionality of RBC can be affected from changes in the roughness, becoming more fragile and susceptible to breakage.

The Good Behavior Game (GBG) is an effective intervention to reduce disruptive behavior. The GBG typically involves immediate stimulus presentation (e.g.., delivery of a token) following disruptions; however, experimenters have also removed tokens contingent upon disruptions. In the present study, we compared the effects of the GBG-stimulus presentation (P) and GBG-stimulus removal (R) on levels of disruptions in a 2nd-grade general education classroom. In addition, we measured student prompts, teacher praise and correctives, and student and teacher preference. The GBG-P and GBG-R versions of the game were similarly effective in reducing disruptions. However, the teacher chose to implement the GBG-R and the majority of students reported a preference for the GBG-R.

Hepatocellular carcinoma (HCC) ranks fifth in occurrence and second in mortality of all cancers. The development of effective therapies for HCC is urgently needed. Anticancer drugs targeted to the liver-specific asialoglycoprotein receptors (ASGPRs) are viewed as a promising potential treatment for HCC. ASGPRs facilitate the recognition and endocytosis of molecules, and possibly vehicles with galactose end groups, by the liver. In this study, bovine serum albumin (BSA) was conjugated with lactose using a thermal treatment. The formation of lactosylated BSA (BSA-Lac) was confirmed by a change of the chemical structure, increased molecular mass, and Ricinus communis lectin recognition. Subsequently, the low-crosslinking BSA-Lac nanoparticles (LC BSA-Lac NPs) and high-crosslinking BSA-Lac nanoparticles (HC BSA-Lac NPs) were synthesized. These nanoparticles presented spherical shapes with a size distribution of 560 &plusmn, 18.0 nm and 539 &plusmn, 9.0 nm, as well as an estimated surface charge of &minus, 26 &plusmn, 0.15 mV and &minus, 24 &plusmn, 0.45 mV, respectively. Both BSA-Lac NPs were selectively recognized by ASGPRs as shown by biorecognition, competition, and inhibition assays using an in vitro model of HCC. This justifies pursuing the strategy of using BSA-Lac NPs as potential drug nanovehicles with selective direction toward hepatocellular carcinoma.

Nanodiamonds when carboxylated (cNDs) act as reducing agents and hence could limit oxidative damage in biological systems. Gamma (γ)-irradiation of whole blood or its components is required in immunocompetent patients to prevent transfusion-associated graft versus host disease (TA-GVHD). However, γ-irradiation of blood also deoxygenates red blood cells (RBCs) and induces oxidative damage, including abnormalities in cellular membranes and hemolysis. Using atomic force microscopy (AFM) and Raman spectroscopy, we examined the effect of cNDs on γ-irradiation mediated deoxygenation and morphological damage of RBCs. γ-Radiation induced several morphological phenotypes, including stomatocytes, codocytes and echinocytes. While stomatocytes and codocytes are reversibly damaged RBCs, echinocytes are irreversibly damaged. AFM images show significantly fewer echinocytes among cND-treated γ-irradiated RBCs. The Raman spectra of γ-irradiated RBCs had more oxygenated hemoglobin patterns when cND-treated, resembling those of normal, non-irradiated RBCs, compared to the non-cND-treated RBCs. cND inhibited hemoglobin deoxygenation and morphological damage, possibly by neutralizing the free radicals generated during γ-irradiation. Thus cNDs have the therapeutic potential to preserve the quality of stored blood following γ-irradiation.

The compounds 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza-17,40-dioxa[10.1.10.1]paracyclophane and 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza[10.1.10.1]paracyclophane binuclear copper complexes (Cu2PO and Cu2PC, respectively) were studied by determining their antioxidant capacity using the TROLOX equivalent antioxidant capacity (TEAC) assay, and their cytotoxicity on cultured cells, as well as the superoxide dismutase (SOD)-like activity. Cu2PO had an antioxidant capacity (0.1 g eq TROLOX mol−1) within the order of magnitude of ascorbic acid, and both, Cu2PO and Cu2PC were nontoxic to cultured peripheral mononuclear blood cells. The SOD-like activity was evaluated using the nitroblue tetrazolium assay, and both compounds presented an excellent activity: for Cu2PO, the IC50 was 52 nM and for Cu2PC an IC50 of 0.5 μM was obtained comparable to CuZn SOD IC50 17 nM (Fernandes et al., J Inorg Biochem 2007;101:849–858). These results suggest that synthetic binuclear macrocycles are good candidates to be used as synthetic bioactive molecules with applications in biomedicine.

Capping of RNA molecules with m7G is an essential modification which plays several roles in RNA metabolism and processing. In the nucleus it directs pre-mRNAs to the processing pathway and mRNAs and U small nuclear RNAs (U snRNAs) to the export pathway. In the cytoplasm, it regulates both mRNA translation initiation and mRNA turnover. In most eukaryotic organisms m7G capping is restricted to nascent RNA polymerase II (PolII) transcripts, namely, pre- mRNAs and precursors to U snRNAs. In contrast, in trypanosomatid protozoa mRNA capping occurs by a fundamentally different mechanism. It is well established that the m7G cap of mature mRNA is acquired posttranscriptionally by an RNA processing reaction, namely, trans splicing. In this process, the capped spliced leader (SL) sequence is transferred from the SL RNA to the 5′ ends of all trypanosome mRNAs (14, 23, 36). Thus, trans splicing can also be considered a trans-capping reaction. The cap structure of the Trypanosoma brucei and Crithidia fasciculata SL RNA is quite elaborate in that the m7G moiety is linked via a 5′-5′ triphosphate bridge to four methylated nucleotides, resulting in an unusual cap 4 structure (2) which is essential for utilization of the SL RNA in trans splicing (18, 43). At present the identity of the RNA polymerase transcribing the SL RNA genes is not defined, because the standard classification based on transcription inhibitors gave conflicting results as to whether PolII or PolIII is the responsible polymerase (8, 25, 26, 42). In addition to the SL RNA, a subset of trypanosome PolIII transcripts, namely, the U2, U3, and U4 snRNAs, is also initially m7G capped (7, 22, 24, 39). Despite the uncertainty about the polymerase transcribing the SL RNA genes, the fact that some PolIII transcripts are capped suggests that the mechanism of transcript selection by the capping enzyme is different in trypanosomes from that in other eukaryotes. m7G capping is mediated by the stepwise action of three enzymatic activities (for a review, see reference 33). First, the γ-phosphate of a primary transcript is removed by RNA 5′-triphosphatase followed by GTP:RNA guanylyltransferase, or capping enzyme, which caps the RNA by the addition of GMP. Finally, the newly attached guanine residue is methylated at the N-7 position by the action of RNA (guanine-7-)methyltransferase. So far, only the reaction mechanism of guanylyltransferase has been examined in some detail. This enzyme catalyzes two sequential nucleotidyl transfer reactions, with a covalent enzyme-guanylate intermediate (19, 30). In this reaction, nucleophilic attack on the α-phosphate of GTP by guanylyltransferase results in the release of pyrophosphate and the formation of a covalent adduct in which GMP is linked to the guanylyltransferase through a phosphoamide bond to the ɛ-amino group of the catalytic lysine residue (30, 34, 38). To complete the reaction, GMP is transferred to the 5′ end of substrate RNA to yield an inverted 5′-5′ triphosphate bond. The vaccinia virus capping enzyme is a multifunctional protein that carries out all three steps of the capping reaction (21, 40, 44). The protein is organized as a heterodimer, with subunits of 95 and 33 kDa (4, 10, 16, 29, 32). The RNA 5′-triphosphatase and guanylyltransferase have been mapped to the amino-terminal 60-kDa domain of the large subunit, while full (guanine-7-)methyltransferase activity requires both subunits. The subunit structure of the vaccinia virus capping enzyme is only partially maintained in cellular counterparts. In particular, cellular capping enzymes are distinct from their viral counterparts in that there is so far no example of a physical association between the capping and methyltransferase functions. In Saccharomyces cerevisiae, the purified capping enzyme consists of two monofunctional polypeptides: a 52-kDa guanylyltransferase and an 80-kDa triphosphatase (12, 13, 28). The guanylyltransferase is the product of the CEG1 gene, which is essential for cell viability (28). As in higher eukaryotes, the yeast (guanine-7-)methyltransferase is purified as a separate entity and is encoded by the ABD1 gene (15). The monofunctional domain structure of the guanylyltransferase is also present in other fungi, such as Schizosaccharomyces pombe (31) and Candida albicans (49), and in Chlorella virus PBCV-1 (11). In higher eukaryotes, biochemical fractionation has shown that the guanylyltransferase from rat liver copurifies with an RNA triphosphatase activity but that the (guanine-7-)methyltransferase readily separates in early chromatography steps and purifies as an unassociated enzyme (48). Recent cloning of the Caenorhabditis elegans (37, 46) and mammalian (17, 50) capping enzymes showed that these enzymes consist of a single bifunctional polypeptide with two domains: a carboxy-terminal guanylyltransferase domain and an amino-terminal domain with RNA triphosphatase activity. Even though the guanylyltransferase domain is strictly conserved with respect to amino acids that are essential for in vivo function, the RNA triphosphatase domain does not resemble the vaccinia virus triphosphatase domain but rather has significant sequence and mechanistic similarities to the family of protein tyrosine phosphatases. Thus, it would appear from the above examples that unicellular and multicellular organisms differ with respect to the physical organization of enzymatic activities of the capping machinery; whereas unicellular organisms have a monofunctional guanylyltransferase, multicellular organisms have a bifunctional triphosphatase-guanylyltransferase. As a first step toward understanding the process of RNA capping in trypanosomes, we have purified the capping enzyme from C. fasciculata and cloned the corresponding genes from C. fasciculata and T. brucei. Comparison of the predicted amino acid sequences of the trypanosome proteins with those of the available eukaryotic and viral capping enzymes revealed several unique structural features. In particular, the trypanosome capping enzymes are remarkable in that they include a novel amino-terminal domain that displays no resemblance to any other domain associated with capping enzymes.

Summary Background & aims Obesity was recognized as an independent risk factor for morbidity and mortality during last influenza A/H1N1 pandemic. Mechanisms involved in the high mortality risk from obesity during influenza A virus include reduced type I interferon production and delayed pro-inflammatory response, which lead to a higher rate of morbidity and mortality in murine models. In this study, we evaluated the production of type I interferons, pro-inflammatory and anti-inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) from obese and lean subjects with and without confirmed infection of influenza A/H1N1. The expression levels of the suppressor of cytokine signaling-1 (SOCS1), SOCS3 and nuclear factor-kB were also evaluated. Methods Cytokines were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and/or by ELISA in PBMCs stimulated with toll like receptor-3 (TLR-3) and TLR-7 ligands. The mRNA expression of SOCS1 and SOCS3 were evaluated by qRT-PCR. Results The obese volunteers infected with influenza A/H1N1 showed a diminished ability to produce type I interferon in response to TLR-3 ligand. Interestingly, the pro-inflammatory response was also affected in TLR-3 stimulated PBMCs. Obese influenza-free volunteers showed an increased basal expression of SOCS3, but not SOCS1. During influenza infection, SOCS1 and SOCS3 expression was higher in the lean infected volunteers in contrast to those who were obese infected. Conclusions These data suggest that obesity is related to TLR-3 impairment and explain, at least in part, the inadequate immune response of obese individuals during infection with influenza A/H1N1 virus.

Swine influenza virus (SwIV) is considered a zoonosis and the fact that swine may act as an intermediate reservoir for avian influenza virus, potentially infectious for humans, highlights its relevance and the need to understand the interaction of different influenza viruses with the porcine immune system. Thus, in vitro porcine bone marrow-derived dendritic cell (poBMDCs) were infected with a circulating SwIV A/Swine/Spain/SF32071/2007(H3N2), 2009 human pandemic influenza virus A/Catalonia/63/2009(H1N1), low pathogenic avian influenza virus (LPAIV) A/Anas plathyrhynchos/Spain/1877/2009(aH7N2) or high pathogenic avian influenza virus (HPAIV) A/Chicken/Italy/5093/1999(aH7N1). Swine influenza virus H3N2 infection induced an increase of SLA-I and CD80/86 at 16 and 24 h post infection (hpi), whereas the other viruses did not. All viruses induced gene expression of NF-κB, TGF-β, IFN-β and IL-10 at the mRNA level in swine poBMDCs to different extents and in a time-dependent manner. All viruses induced the secretion of IL-12 mostly at 24 hpi whereas IL-18 was detected at all tested times. Only swH3N2 induced IFN-α in a time-dependent manner. Swine H3N2, aH7N2 and aH7N1 induced secretion of TNF-α also in a time-dependent manner. Inhibition of NF-κB resulted in a decrease of IFN-α and IL-12 secretion by swH3N2-infected poBMDC at 24 hpi, suggesting a role of this transcription factor in the synthesis of these cytokines. Altogether, these data might help in understanding the relationship between influenza viruses and porcine dendritic cells in the innate immune response in swine controlled through soluble mediators and transcription factors., This work was partly funded by the Project No. CSD 2006-00007, AGL2006-13809-C03-01, AGL2009-12945-C02-01 and AGL2010-22200-C02-01 by the Spanish Government. PhD studies of Mrs. Tufária Mussá and Masssimiliano Baratelli are funded by doctoral grants from the AECID and MICIN respectively.