M2 Sequence Research Articles

Advancing siRNA Therapeutics targeting MCT-4: A Multifaceted approach integrating Arithmetical Designing, Screening, and molecular dynamics validation.

Monocarboxylate transporter 4 (MCT-4) is involved in various metabolic processes which are crucial in maintaining cellular pH and energy metabolism, and thus influence the tumor microenvironment. The study is aimed to rationally design effective Small interfering RNA (siRNA) that can silence MCT-4. We utilized a comprehensive workflow integrating multiple tools such as siDirect version 2.0, Oligowalk and i-score designer, to evaluate sequence features and predict target site accessibility, Guanine-Cytosine (GC) content and thermodynamic stability. Five (M1, M2, M3, M4 and M5) siRNAs sequences were retrived and subjected to further scrutiny on the account of off-target elimation, sequence conservation, secondary structure formation, and thermodynamic properties. The M1 demonstrated off targets and the M2 sequence showed secondry conformation and therefore M3, M4 and M5 were considered for further evaluation. Additionally, molecular docking and simulations (50ns) were conducted with human Argonaute 2 protein (h-Arg-2). The post- molecular dynamics (MD) analysis revealed M4 (5'UUGAAGAAGACACUGACGG3') as a most appropriate siRNA candidate agsint MCT-4 on the basis of Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and H-Bond results. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis was also performed to further validate the selected siRNA candidates, which further affirmed M4 (5'UUGAAGAAGACACUGACGG3') as an potential candidate for future in-vitro and in-vivo evaluation.

ESR/U-series dating of palaeontological remains from the Neanderthal site of Mutzig-Rain (Alsace, France)

The Middle Paleolithic site of Mutzig-Rain, discovered in 1992, has been studied through several pits (called M1 to M15) and excavation area since 2009, evidencing several localities and delivering more than 5000 lithic artifacts and 12,000 well preserved paleontological remains. The main excavation M2 sequence corresponds to a collapsed rock shelter, containing several levels of Middle Paleolithic occupation. The faunal assemblage of the main archaeological levels (l.7a to 7c) from this sequence includes woolly mammoth, reindeer, horse, bison and woolly rhinoceros, reflecting both cold climate and a steppe context for the whole sequence, while microfauna from the lower levels (l.7d and l.10) indicate the occasional presence of forests that could correspond to a cooler climate. In 1995, U-series analyses on bones from M8 were attempted but the obtained results did not follow the sample stratigraphic order. Later, an OSL dating study has placed the M2 archaeological sequence as coeval to the onset of the Weichselian glacial stage (MIS5d-5a), between 100 and 80 ka.The good preservation of the abundant faunal remains at Mutzig-Rain has allowed their use for ESR/U-series dating studies and the results obtained on Mutzig horse teeth are given in the present paper. A first set of analyses of teeth carried out from the M2 excavation provided ages around 106-96 ka for levels M2 l.7, in agreement with the OSL data, despite quite systematic evidence of U-leaching from the dental tissues and the consequent use of the Accelerating Uptake (AU) model for the age calculation. A second set of analyses on teeth from the M2 and M8 localities confirms this uranium leaching trend, placing the deposition of these levels ca 133 -122 ka.Taking the whole set of ESR/U-series ages, which are slightly older than the OSL dates, would suggest that the stratigraphic sequence was deposited between ca 130 ka (M8 l. 6 and M2 l.10) and ca 100 ka (M2 l.7), i.e. on a time range covering the Eemian Interglacial (MIS5e) and the subsequent Early Weichselian Glacial stage (MIS5d-5a). However, as the faunal record indicates a cool climate and an open steppe-like environment for the whole archaeological sequence, the ESR/U-series dating to the Eemian Interglacial in the Mutzig-Rain sequence seems unlikely. In order to check the quality of the paleodosimetric reconstruction of the ESR/U-series analyses, an additional isochron study was performed for the two levels for which several teeth were analyzed: M2 l.7 (n = 4) and M8 l.6 (n = 3). This isochron study validates the proposed reconstruction and confirms the Early Weichselian age of these two levels. These results confirm the chronology established previously with OSL and places Mutzig-Rain as a reference site for the understanding of the Neanderthal Middle Palaeolithic occupation in Eastern France.

An experimental universal swine influenza a virus (IAV) vaccine candidate based on the M2 ectodomain (M2e) peptide does not provide protection against H1N1 IAV challenge in pigs

Swine flu is a common disease problem in North American pig populations and swine influenza A viruses (IAV) are extremely diverse and the lack of cross protection between heterologous strains is impacting vaccine efficacy in the field. The objective of this study was to design and test a novel swine flu vaccine targeting the M2 ectodomain (M2e) of IAV, a highly conserved region within the IAV proteome. In brief, an M2e peptide was designed to match the predominant swine IAV M2 sequence based on global analysis of sequences from pigs and humans. The resulting sequence was used to synthesize the M2e peptide coupled to a carrier protein. The final vaccine concentration was 200 µg per dose, and a commercial, microemulsion-based aqueous adjuvant was added. Nine 3-week-old IAV negative piglets were randomly assigned to three groups and rooms including non-vaccinated pigs (NEG-CONTROLs) and vaccinated pigs using the intramuscular (M2e-IM) or the intranasal route (M2e-IN). Vaccinations were done at weaning and again at 2 weeks later. An in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated to study the M2e IgG antibody response and demonstrated M2e-IM pigs had a higher systemic antibody response compared to M2e-IN pigs. Subsequently, an IAV challenge study was conducted. The results indicated that M2e-IM vaccinated pigs were not protected from H1N1 (US pandemic clade, global clade 1A.3.3.2) challenge despite having a strong humoral anti-M2e immune response. In conclusion, while the experimental IAV vaccine was able to induce anti-M2e antibodies, when challenged with H1N1, the vaccinated pigs were not protected, perhaps indicating that reactivity to the M2e antigen alone is not sufficient to reduce clinical signs, lesions or shedding associated with experimental IAV challenge.

Coexistence of single-particle and collective excitation in Ni61

The high spin states in 61 Ni have been studied using the fusion evaporation reaction, Ti( $^{14}$C,3n) $^{61}$Ni at an incident beam energy of 40 MeV. A Compton suppressed multi-HPGe detector setup, consisting of six Clover detectors and three single crystal HPGe detectors was used to detect the de-exciting $\gamma$ rays from the excited states. The level scheme has been extended up to an excitation energy of 12.8 MeV and a tentative J$_{\pi}$ = 35/2$^+$ . The low-lying negative parity levels are found to be generated by single particle excitation within the f p shell and also excitations to the g$_{9/2}$ orbitals as explained well with shell model calculations using the GXPF1Br+V M U (modified) interaction. Two rotational structure of regular E2 sequences with small to moderate axial deformation have been established at higher excitation energy. Most interestingly, two sequences of M1 transitions are reported for the first time and described as magnetic rotational bands. The shears mechanism for both the bands can be described satisfactorily by the geometrical model. The shell model calculation involving the cross shell excitation beyond the fp shell well reproduce the M1 and E2 sequences. The shell model predicted B(M1) values for the magnetic rotational band B1 show the decreasing trend with spin as expected with closing of the shears.

A Glu-Glu-Tyr Sequence in the Cytoplasmic Tail of the M2 Protein Renders Influenza A Virus Susceptible to Restriction of the Hemagglutinin-M2 Association in Primary Human Macrophages.

Influenza A virus (IAV) assembly at the plasma membrane is orchestrated by at least five viral components, including hemagglutinin (HA), neuraminidase (NA), matrix (M1), the ion channel M2, and viral ribonucleoprotein (vRNP) complexes, although particle formation is observed with expression of only HA and/or NA. While these five viral components are expressed efficiently in primary human monocyte-derived macrophages (MDMs) upon IAV infection, this cell type does not support efficient HA-M2 association and IAV particle assembly at the plasma membrane. Both defects are specific to MDMs and can be reversed upon disruption of F-actin. However, the relationship between the two defects is unclear. Here, we examined whether M2 contributes to particle assembly in MDMs and if so, which region of M2 determines the susceptibility to the MDM-specific and actin-dependent suppression. An analysis using correlative fluorescence and scanning electron microscopy showed that an M2-deficient virus failed to form budding structures at the cell surface even after F-actin was disrupted, indicating that M2 is essential for virus particle formation at the MDM surface. Notably, proximity ligation analysis revealed that a single amino acid substitution in a Glu-Glu-Tyr sequence (residues 74 to 76) in the M2 cytoplasmic tail allowed the HA-M2 association to occur efficiently even in MDMs with intact actin cytoskeleton. This phenotype did not correlate with known phenotypes of the M2 substitution mutants regarding M1 interaction or vRNP packaging in epithelial cells. Overall, our study identified M2 as a target of MDM-specific restriction of IAV assembly, which requires the Glu-Glu-Tyr sequence in the cytoplasmic tail. IMPORTANCE Human MDMs represent a cell type that is nonpermissive to particle formation of influenza A virus (IAV). We previously showed that close proximity association between viral HA and M2 proteins is blocked in MDMs. However, whether MDMs express a restriction factor against IAV assembly or whether they lack a dependency factor promoting assembly remained unknown. In the current study, we determined that the M2 protein is necessary for particle formation in MDMs but is also a molecular target of the MDM-specific suppression of assembly. Substitutions in the M2 cytoplasmic tail alleviated the block in both the HA-M2 association and particle production in MDMs. These findings suggest that MDMs express dependency factors necessary for assembly but also express a factor(s) that inhibits HA-M2 association and particle formation. High conservation of the M2 sequence rendering the susceptibility to the assembly block highlights the potential for M2 as a target of antiviral strategies.

Intra‐tooth isotopic analysis (δ13C and δ15N) of dentine collagen in high‐crowned teeth: A new experimental study with modern sheep specimens

AbstractHypsodont teeth molars develop in a short lapse of time. Both dentine and enamel dental tissues do not regenerate once formed. This fact allows the reconstruction of isotopic sequences covering short moments of the individual's life span, a useful approach in current zooarchaeological and paleontological studies. Sampling design usually follows a serial or sequential extraction of dental bands perpendicular to the tooth growth axis, from the apex to the root. However, dentine develops following a secretion of layers oblique to the tooth vertical axis, and this pattern makes hard to isolate isotopic signals. A new experimental study with modern sheep breeds was conducted, covering a whole year and where carbon and nitrogen isotopic signatures of consumed plants were known. Three diet periods with different isotopic compositions were alternated during sheep's life. In this contribution, we test the resolution of a dentine sampling procedure to obtain intra‐tooth isotopic variability of δ13Ccollagen and δ15Ncollagen values tied to diet shifts in sheep. Dentine was sequentially sampled in previously demineralized second and third mandibular molars. Samples were treated with standard protocols of collagen purification and then subjected to stable carbon and nitrogen isotope analysis. In M2 and M3 isotopic sequences obtained, diet shifts are recorded along two progressive trends of increasing–decreasing values, but some affections of the sampling procedure are also present. We conclude that the dentine sequential sampling here implemented allows for obtaining reliable intra‐tooth isotopic data tied to dietary conditions in high‐crowned teeth, although dentine growth patterns should be considered in detail in final interpretations. The integration of this high‐resolution analysis in archaeological studies offers a new scope of possibilities for studying dietary patterns, seasonal dietary changes, and animal husbandry practices in past populations.

HA and M2 sequences alter the replication of 2013–16 H1 live attenuated influenza vaccine infection in human nasal epithelial cell cultures

From 2013 to 2016, the H1N1 component of live, attenuated influenza vaccine (LAIV) performed very poorly in contrast to the inactivated influenza vaccine. We utilized a primary, differentiated human nasal epithelial cell (hNEC) culture system to assess the replication differences between isogenic LAIVs containing the HA segment from either A/Bolivia/559/2013 (rBol), which showed poor vaccine efficacy, and A/Slovenia/2903/2015 (rSlov), which had reasonable vaccine efficacy. There were minimal differences in infectious virus production in Madin-Darby Canine Kidney (MDCK) cells, but the rSlov LAIV showed markedly improved replication in hNEC cultures at both 32 °C and 37 °C, demonstrating that the HA segment alone could impact LAIV replication in physiologically relevant systems. The rSlov-infected hNEC cultures showed stronger production of interferon and proinflammatory chemokines which might also be contributing to the increased overall vaccine effectiveness through enhanced recruitment and activation of immune cells. An M2-S86A mutation had no positive effects on H1 LAIV replication in hNEC cultures, in contrast to the increased infectious virus production seen in an H3 LAIV. No obvious defects in viral RNA packaging were detected, suggesting that HA function, rather than defective particle production, may be driving the differential infectious virus production in hNEC cultures. Overall, we have shown that not all H1 HA segments can be successfully used in LAIV, and this phenotype cannot be fully explained by segment incompatibilities. Physiologically relevant temperatures and primary cell cultures should be used to demonstrate that candidate LAIVs can replicate efficiently, which is a necessary property for effective vaccines.

Virtual Screening Identifies Chebulagic Acid as an Inhibitor of the M2(S31N) Viral Ion Channel and Influenza A Virus.

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.

Dissociable implicit sequence learning mechanisms revealed by continuous theta-burst stimulation.

The primary motor area (M1) has been implicated in visuomotor sequence learning. However, it has been suggested there are multiple neural networks that undertake visuomotor sequence learning. The role of M1 in sequence learning may be specific to learning simple sequences comprising predictable associations between adjacent movements. This study aimed to investigate the role of M1 in learning simple ("first-order conditional") and more complex ("second-order conditional") sequences. It was hypothesized that continuous theta burst stimulation (cTBS) over M1 would result in poorer learning of the simple sequence only. Forty-eight healthy adults received cTBS to either M1 or the parietal lobe or received sham cTBS before immediately completing 2 visuomotor sequence learning tasks. The tasks only differed in relation to the structure (i.e., simple vs. complex) of the sequence. The group who received cTBS over M1 demonstrated significantly poorer learning of the simple sequence in comparison to the more complex sequence. The parietal lobe stimulation and sham stimulation did not affect learning of either sequence. This is the first study to show differential involvement of M1 in visuomotor sequence learning, dependent on sequence structure. The study provides new evidence that sequence learning might be supported by different networks in the brain. Specifically, M1 sequence learning appears to be important for learning simple item-to-item associations but not for more complex sequences. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Deep Mutational Scan of the Highly Conserved Influenza A Virus M1 Matrix Protein Reveals Substantial Intrinsic Mutational Tolerance.

Influenza A virus matrix protein M1 is involved in multiple stages of the viral infectious cycle. Despite its functional importance, our present understanding of this essential viral protein is limited. The roles of a small subset of specific amino acids have been reported, but a more comprehensive understanding of the relationship between M1 sequence, structure, and virus fitness remains elusive. In this study, we used deep mutational scanning to measure the effect of every amino acid substitution in M1 on viral replication in cell culture. The map of amino acid mutational tolerance we have generated allows us to identify sites that are functionally constrained in cell culture as well as sites that are less constrained. Several sites that exhibit low tolerance to mutation have been found to be critical for M1 function and production of viable virions. Surprisingly, significant portions of the M1 sequence, especially in the C-terminal domain, whose structure is undetermined, were found to be highly tolerant of amino acid variation, despite having extremely low levels of sequence diversity among natural influenza virus strains. This unexpected discrepancy indicates that not all sites in M1 that exhibit high sequence conservation in nature are under strong constraint during selection for viral replication in cell culture.IMPORTANCE The M1 matrix protein is critical for many stages of the influenza virus infection cycle. Currently, we have an incomplete understanding of this highly conserved protein's function and structure. Key regions of M1, particularly in the C terminus of the protein, remain poorly characterized. In this study, we used deep mutational scanning to determine the extent of M1's tolerance to mutation. Surprisingly, nearly two-thirds of the M1 sequence exhibits a high tolerance for substitutions, contrary to the extremely low sequence diversity observed across naturally occurring M1 isolates. Sites with low mutational tolerance were also identified, suggesting that they likely play critical functional roles and are under selective pressure. These results reveal the intrinsic mutational tolerance throughout M1 and shape future inquiries probing the functions of this essential influenza A virus protein.

Prefrontal transcranial alternating current stimulation improves motor sequence reproduction

Cortical activity in frontal, parietal, and motor regions during sequence observation correlates with performance on sequence reproduction. Increased cortical activity observed during observation has therefore been suggested to represent increased learning. Causal relationships have been demonstrated between M1 and motor sequence reproduction and between parietal cortex and bimanual learning. However, similar effects have not been reported for frontal regions despite a number of reports implicating its involvement in encoding of motor sequences. Investigating causal relations between cortical activity and reproduction of motor sequences in parietal, frontal and primary motor regions can disentangle whether specific regions during simple observation can be selectively ascribed to encoding or reproduction or both. We designed a sensorimotor paradigm that included a strong motor sequence component, and tested the impact of individually adjusted transcranial alternating current stimulation (IAF-tACS) to prefrontal, parietal, and primary motor regions on electroencephalographic motor rhythms (alpha and beta bandwidths) during motor sequence observation and the ability to reproduce the observed sequences. Independently of the stimulated region, IAF-tACS led to a reduction in suppression in the lower beta-range relative to sham. Prefrontal IAF-tACS however, led to significant improvement in motor sequence reproduction, pinpointing the crucial role of prefrontal regions in motor sequence reproduction.

Permanent tooth emergence: Timing and sequence in a sample of Black Southern African children.

This study investigates mean age, sequence, and temporal trends of permanent tooth emergence in Black Southern African children and compares the findings with other population samples. This community-based cross-sectional study involved 639 Black Southern African children between 5 and 20 years of age. Probit analysis was used to derive the mean age at emergence of the permanent teeth. Sex and cross-population comparisons were undertaken to determine similarities and differences in emergence timing and sequence. Females emerged all teeth earlier except for M3s (p < .05). Black Southern Africans have earlier mean ages of emergence compared to population samples from the USA, Europe, Australia, and Asia. Sexual dimorphism was detected in the mandibular I1/M1 emergence sequence (females, M1 I1 ; males, I1 M1 ). The sequence in males is similar in both jaws to males from other sub-Saharan African, USA, and European samples. Females show a similar sequence pattern in the maxilla with other sub-Saharan African, and also Australian and US females of European ancestry. There is a high frequency of polymorphism in the P1P2C1 emergence sequence, with significantly more P2P1C1 maxillary sequences seen among males. Polymorphic variation was common for the I1 M1 sequence in both males and females. Mean age of tooth emergence among Black Southern African children is similar to children from most other sub-Saharan African populations. No temporal change was seen in the mean age of emergence. Earlier permanent tooth emergence in Black Southern Africans is part of a general sub-Saharan pattern that is distinct from European and Asian populations.

Antiviral resistance markers in influenza virus sequences in Mexico, 2000-2017.

BackgroundInfluenza causes high rates of morbidity and mortality. Genetic variability of influenza viruses generates resistance to antivirals, which are of two types, since they act on two different viral targets: adamantanes, which block the M2 ion channel, and the neuraminidase (NA) inhibitors.MethodsIn Mexico, the available studies on the antiviral resistance of circulating influenza strains are scarce, so this work undertook an analysis of the Mexican sequences reported in public gene banks to perform a systematic analysis of the antiviral resistance markers on both M2 and NA. In all, 284 M2 sequences and 423 NA sequences were retrieved from three genetic databases (sequences from 2000 to 2017 were considered).ResultsThe resistance markers to M2 blockers were present in 100% of H1N1 pdm2009, 83.6% of H3N2, and 5.8% of seasonal H1N1 sequences. Two resistance markers conferring resistance to NA inhibitors were present in seasonal H1N1 sequences, H275Y (50.0%) and N70S (33.3%). None of these viruses had both resistance markers, which are associated with oseltamivir resistance. The more frequent resistance marker in H1N1 pdm2009 NA sequences was H275Y, present in 3.6%, while S247N was present in 0.30%. Only one of the resistance-associated markers (Q136K) in NA (1.5%) was present in the analyzed H3N2 sequences, while sequences of influenza B virus did not present resistance markers to NA inhibitors. Some influenza A H1N1 pdm2009 sequences (1.8%) presented resistance markers to both M2 and NA.ConclusionBased on the present analysis, 7.1% of the all serotypes of influenza virus A sequences analyzed in Mexico from 2000 to 2017 have mutations conferring resistance to NA inhibitors. Because of this, and the limited availability of influenza drugs, it is necessary to increase the epidemiological surveillance, including molecular analysis, which will provide data such as the presence of changes associated with antiviral resistance.

History of matrix genes mutations within PCR target regions among circulating influenza H3N2 clades over ten-plus-years

BackgroundEmerging influenza A/H3N2 clades have been associated with M1 gene mutations which affect the performance of commercial PCR assays. Objectives and study designThe evolution and prevalence of problematic M1 mutations, and their associated viral clades, were investigated. All European and USA isolates from the GISAID database with both HA and M1 sequences available, collected during the respiratory seasons from the Fall of 2007 through January of 2018, were analyzed. ResultsFive M1 target region patterns, designated A–E, were observed in more than 10% of the isolates during a season, with patterns that appeared sequentially, each having one additional mutation. The C153T mutation was universal. Pattern A, which only had the single mutation, predominated between 2007/08 and 2009/10. Dual- and triple-mutation patterns (B and C) emerged in 2010/11 and 2011/12 respectively, and pattern C predominated for one season (2012/13). In 2012/13, the problematic quadruple-mutation containing C163T first appeared in 3C.2 viruses. Seasons 2013/14 and 14/15 were associated with significant viral diversity with five clades and four M1 patterns co-circulating, with different rates in Europe and the USA. Since 2014, clade 3C.2a with M1 pattern D has emerged as the predominant type. During 2016/17 season, a new quintuplet mutation pattern (E) emerged in cluster 3C.2a1 isolates. ConclusionsM1 target region mutations have been prevalent for more than ten years, with the number of mutations continually increasing. Often population inferences of M1 mutations can be made based on viral clade. However, gene segment reassortment can affect predictive abilities.

Stability and donor-acceptor bond in dinuclear organometallics CpM1-M2Cl3 (M1, M2 = B, Al, Ga, In; Cp = η 5-C5H5).

The geometries and stabilities of the dinuclear organometallics CpM1-M2Cl3 (M1, M2 = B, Al, Ga, In; Cp = η 5-C5H5) have been investigated by density functional theory (DFT) at M06L/6-311G(d, p) levels. The nature of the donor-acceptor M1 → M2 bond was also studied based on the atoms in molecules (AIM) theory, energy decomposition analysis (EDA) and natural bond orbital (NBO) analysis. The results show that the electronegativity of the M atom determines the stability and covalent character of the dinuclear organometallics CpM1-M2Cl3. The compounds in which the M with larger electronegativity acts as the donor are more stable than in those in which it acts as the acceptor in the donor-acceptor bond, and the donor-acceptor bond has more covalent characteristics. The strength and polarity of the M1 → M2 donor-acceptor bond is determined by the periodicity of the M atom. When the period number of the M1 atom is smaller than that of M2, the strength of the M1 → M2 bond is larger than that of the M2 → M1 bond. For homonuclear dinuclear organometallics, the polarity of the M-M bond increases with increasing atomic number of the M atom. For heteronuclear complexes, the polarity of the M1-M2 bond for a given M1 also increases in the sequence of M2 = B, Al, Ga, and In. Graphic abstract Molecular graph and electron location function isosurfaces map of CpM 1 -M 2 Cl 3(small red spheres represent bond critical points).

A single dose and long lasting vaccine against pandemic influenza through the controlled release of a heterospecies tandem M2 sequence embedded within detoxified bacterial outer membrane vesicles.

The influenza A virus undergoes genetic drift and shift, leaving the general population susceptible to emerging pandemic strains, despite seasonal flu vaccination. Here we describe a single dose influenza vaccine derived from recombinant outer membrane vesicles (rOMVs) that display an antigen-mapped heterospecies tandem sequence of the M2 protein from the influenza A virus, released over 30days from poly(lactic-co-glycolide) (PLGA) microparticles. Four weeks post vaccination, BALB/c mice developed high anti-M2e IgG titers that were equivalent to those generated at 8weeks in a typical prime/boost vaccine regimen. Challenge of mice with a lethal dose of mouse adapted influenza virus PR8 (H1N1) 10weeks post vaccination resulted in 100% survival for both rOMV single-dose microparticle and prime/boost vaccinated mice. Anti-M2e IgG1 and IgG2a antibody titers were weighted toward IgG1, but splenocytes isolated from rOMV single-dose microparticle vaccinated mice produced high levels of IFNγ relative to IL-4 in response to stimulation with M2e peptides, supporting a more Th1 biased immune response. The protective immune response was long lasting, eliciting sustained antibody titers and 100% survival of mice challenged with a lethal dose of PR8 six months post initial vaccination. Together, these data support the potential of controlled release rOMVs as an effective single dose, long lasting and rapidly effective vaccine to protect against influenza.

Myeloid HIFs are dispensable for resolution of inflammation during skeletal muscle regeneration.

Besides their role in cellular responses to hypoxia, hypoxia-inducible factors (HIFs) are involved in innate immunity and also have anti-inflammatory (M2) functions, such as resolution of inflammation preceding healing. Whereas the first steps of the inflammatory response are associated with proinflammatory (M1) macrophages (MPs), resolution of inflammation is associated with anti-inflammatory MPs exhibiting an M2 phenotype. This M1 to M2 sequence is observed during postinjury muscle regeneration, which provides an excellent paradigm to study the resolution of sterile inflammation. In this study, using in vitro and in vivo approaches in murine models, we demonstrated that deletion of hif1a or hif2a in MPs has no impact on the acquisition of an M2 phenotype. Furthermore, using a multiscale methodological approach, we showed that muscles did not require macrophagic hif1a or hif2a to regenerate. These results indicate that macrophagic HIFs do not play a crucial role during skeletal muscle regeneration induced by sterile tissue damage.

Investigation of a recent rise of dual amantadine-resistance mutations in the influenza A M2 sequence

BackgroundThe S31N amantadine-resistance mutation in the influenza A M2 sequence currently occurs more frequently in nature than the S31 wild type. Overcoming the resistance of the S31N mutation is the primary focus of M2 researchers who aim to develop novel antiviral therapies. Recent studies have noted a possible rise in frequency of the V27A/S31N double amantadine-resistance mutation in recent years. The purpose of this study is to investigate this recent rise in frequency of the double mutation and any possible bias of the other mutations toward co-occurrence with S31N or S31 strains.ResultsThe primary dataset used for this study was comprised of 24,152 influenza A M2 channel sequences which were downloaded from UniProt. There is an increased frequency for the S31N/V27A dual AR mutation in recent years, especially in swine. A test for difference in two proportions indicates that the V27A mutation is co-occurring with S31N more often than expected (p-value < 0.001) when considering individual amino acid frequencies. At the same time, the different propensities for the V27A as compared to the V27T dual mutant may reflect differences in viral fitness or protein energetics, and this information could be exploited to focus drug development so as to reduce further drug insensitivity.ConclusionsThe development of the S31N/V27A variant in the Midwestern US swine may be a harbinger of novel human strain development. V27A/S31N is a possible path forward for the evolution of M2 which may convey a new level of drug resistance and should receive attention in drug design.

Effects of muscimol inactivations of functional domains in motor, premotor, and posterior parietal cortex on complex movements evoked by electrical stimulation

Parietal and frontal cortex are central to controlling forelimb movements. We previously showed that movements such as reach, grasp, and defense can be evoked from primary motor (M1), premotor (PMC), and posterior parietal (PPC) cortex when 500-ms trains of electrical pulses are delivered via microelectrodes. Stimulation sites that evoked a specific movement clustered into domains, which shared a topographic pattern in New World monkeys and prosimian galagos. Matched functional domains in parietal and frontal cortex were preferentially interconnected. We reasoned that matched functional domains form parallel networks involved in specific movements. To test the roles of domains in M1, PMC, and PPC, we systematically inactivated with muscimol domains in one region and determined if functional changes occurred in matching domains in other regions. The most common changes were higher current thresholds for stimulation-evoked movements and shorter, not fully developed, trajectories of movements. Inactivations of an M1 functional domain greatly reduced or abolished movements evoked from the matching domains in PMC or PPC, whereas movements evoked from nonmatching domains remained mostly unaffected. In contrast, inactivating PMC or PPC domains did not consistently abolish the ability to evoke movements from matching M1 domains. However, inactivation of PMC domains suppressed or altered the movements evoked from PPC domains. Thus movement sequences evoked from PMC depend on M1 and movement sequences evoked from PPC depend on both M1 and PMC. Overall, the results support the conclusion that PPC, PMC, and M1 are subdivided into functional domains that are hierarchically related within parallel networks.

Variability in minimal genomes: Analysis of tandem repeats in the microsporidia Encephalitozoon intestinalis

Microsporidia are ubiquitous fungi with genomes that have undergone a strong reduction to the extreme cases of Encephalitozoon cuniculi and Encephalitozoon intestinalis. Genetic variability within species of the Encephalitozoon genus has been reported, with most of the studies based on the internal transcribed spacer (ITS) of the rDNA. However, in contrast to the picture of E. cuniculi and Encephalitozoon hellem, where different strains have been identified, no genetic variability has yet been observed in E. intestinalis. We have analysed tandem repeats included in putative coding sequences which could be used as polymorphic markers in E. intestinalis. Eight candidate loci (M2, M2A, M3, M5, M7, M7A, M8 and PTP1) were established and 9 E. intestinalis cultured strains from North America, South America and Europe were analysed. M2, M7 and PTP1 nucleotide sequences were identical among the different strains and the GenBank sequence. In contrast, we observed variants in 4 markers (M2A, M3, M7A and M8) which did not correspond to their respective reference sequences. The most noticeable finding was that with the M5 marker two genotypes were defined among the different strains studied, demonstrating genotypic variability of E. intestinalis. Although the diversity described is certainly not high, which can be explained by a lower chance of genetic variability in its minimal genome, we have demonstrated that polymorphisms actually exist in E. intestinalis. Epidemiological studies using this genetic marker should now be conducted to elucidate the genetic variability in E. intestinalis and improve our knowledge of the epidemiology of this microsporidia.