Analogous Substitution Research Articles

Extending the metal to insulator transitions of rare‐earth nickelates towards low temperature ranges

AbstractAlthough the d‐band correlated rare‐earth nickelates (ReNiO3) exhibit broadly adjustable metal to insulator transitions (MIT) that enables emerging correlated electronic applications, it is yet difficult to regulate their associated critical temperature (TMIT) below 100 K. Herein, we extend the lower limit in TMIT of ReNiO3 down to 83 K while maintaining an abrupt switch in resistivity via partial La‐substitution of PrNiO3. The near edge X‐ray fine structure analysis and density function theory calculations indicate the strengthening in the metallic orbital configuration and reduction in the ground state band gap via La‐substitution of Re in NdNiO3 and PrNiO3. In contrast, analogous Ce substitution cannot reduce the TMIT of ReNiO3 owing to its valance variability toward +4, while La partial substitution of ReNiO3 with middle or heavy rare‐earth (e.g., Sm) easily disturb the co‐occupation of the Re‐site by the two rare‐earths as one dispersive phase.

Binding motif for RIC-3 chaperon protein in serotonin type 3A receptors.

Serotonin or 5-hydroxytryptamine type 3 (5-HT3) receptors belong to the family of pentameric ligand-gated ion channels (pLGICs) that are therapeutic targets for psychiatric disorders and neurological diseases. Due to structural conservation and significant sequence similarities of pLGICs' extracellular and transmembrane domains, clinical trials for drug candidates targeting these two domains have been hampered by off-subunit modulation. With the present study, we explore the interaction interface of the 5-HT3A subunit intracellular domain (ICD) with the resistance to inhibitors of choline esterase (RIC-3) protein. Previously, we have shown that RIC-3 interacts with the L1-MX segment of the ICD fused to maltose-binding protein. In the present study, synthetic L1-MX-based peptides and Ala-scanning identify positions W347, R349, and L353 as critical for binding to RIC-3. Complementary studies using full-length 5-HT3A subunits confirm that the identified Ala substitutions reduce the RIC-3-mediated modulation of functional surface expression. Additionally, we find and characterize a duplication of the binding motif, DWLR…VLDR, present in both the MX-helix and the transition between the ICD MA-helix and transmembrane segment M4. Analogous Ala substitutions at W447, R449, and L454 disrupt MAM4-peptide RIC-3 interactions and reduce modulation of functional surface expression. In summary, we identify the binding motif for RIC-3 in 5-HT3A subunits at two locations in the ICD, one in the MX-helix and one at the MAM4-helix transition.

Triptycene-Based Self-Assembled Monolayer as a Template for Successive Click Reactions

Triptycene-based molecules represent a promising platform for tripodal monomolecular assembly on solid substrates. The versatility of the approach can be increased significantly if a decoration of the assembled tripods by purpose-specific functional groups will be possible. In this context, the possibility of click reaction between the 10-ethynyl-substituted 1,8,13-trimercaptomethyltriptycene monolayer (EtTripS) on Au(111) and an azide-decorated substituent is tested and additionally verified by a subsequent derivatization reaction. The primary monolayer is shown to exhibit dense molecular packing, tripodal adsorption geometry, and high orientational order, which qualify it well as a template for subsequent reactions. The characteristic spectroscopic fingerprints of the click and derivatization reactions were recorded. The overall impact of these reactions on the work function of the EtTripS-engineered substrate─used as a measure of their efficiency and as an example of functional properties─was well comparable with the effect of the analogous substitution (−CF3) for monodentate monolayers, emphasizing the high potential of the on-surface modification approach.

Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis.

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203–205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral ‘RG’ motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2’s continued adaptation to human infection.

The analogue substitution model: Introducing competition in the absence of generic substitution in Danish hospitals

Increasingly advanced medicines have created monopolies in treatment areas with no viable options for generic substitution due to patent protection. As health care systems are increasingly under pressure to deliver health care improvements, costs become prohibitive as budgets are under pressure. To create financial flexibility, Danish regional authorities have over the period from 2009 until today developed, implemented, and gradually increased the use of a new model for analogue medicine substitution. The model introduces competitive tenders among patented medicines by declaring these medicines therapeutically equivalent in the treatment of specific diseases and thereby creating new opportunities for reducing annual medicine expenses. The model is based on enhanced collaboration among the health technology assessment (HTA) body, the procurement body, and the hospital owner, and it effectively covers medicine expenditure and cost-effectiveness while ensuring standardized treatments across hospitals and regions. The model is internationally unique in integrating several healthcare stakeholders and balancing their respective agendas to generate optimized outcomes for the healthcare system as a whole. However, some challenges persist, as the HTA process is resource and time consuming. Future research can show whether there are other challenges connected with the model.

Reactivity of a Molecular Calcium Hydride Cation ([CaH]+) Supported by an NNNN Macrocycle.

The hydride ligand in the cationic calcium hydride supported by a NNNN-type macrocycle, [(Me4TACD)2Ca2(μ-H)2(THF)][BAr4]2 (1; Me4TACD = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane; THF = tetrahydrofuran; BAr4 = B(C6H3-3,5-Me2)4), shows, in addition to its Brönsted basicity toward weak acids, a pronounced nucleophilicity resulting in nucleophilic substitution or insertion (addition) at a silicon or sp2 carbon center. Terminal acetylenes RC≡CH (R = SiMe3, cyclopropyl) as well as 1,4-diphenylbutadiene were deprotonated by 1 to give dinuclear complexes [(Me4TACD)2Ca2(μ-C≡CR)2][BAr4]2 (2a, R = SiMe3; 2b, R = cyclopropyl) and [(Me4TACD)2Ca2(μ2-η4-1,4-Ph2C4H2)][BAr4]2 (3) with H2 evolution. The addition reaction with BH3(THF) gave a tetrahydridoborate complex, [(Me4TACD)Ca(BH4)(THF)2][BAr4] (4), with κ2-H2BH2 coordination in the solid state, suggesting a pronounced Lewis acidic calcium center. The behavior resulting from both Lewis acidity and hydricity becomes apparent in the nucleophilic substitution of fluorobenzene by 1 to give benzene and the dimeric fluoride complex [(Me4TACD)2Ca2(μ-F)2(THF)][BAr4]2·2.5THF (5). Analogous nucleophilic substitution reaction is observed for heterofunctionalized organosilanes XSiR3 [X = I, N(SiHMe2)2, N3; R = Me3 or HMe2], which resulted in the formation of calcium complexes [(Me4TACD)Ca(X)(THF)n][BAr4] (6-8) containing an X ligand along with hydrosilane HSiR3. An insertion reaction by 1 was observed with CO2 and CO to give dinuclear formato complex [(Me4TACD)2Ca2(μ-OCHO)2][BAr4]2 (9) and cis-enediolato complex [(Me4TACD)2Ca2(μ-OCH═CHO)][BAr4]2·3.5THF (10), respectively. The latter is believed to have been formed as a result of the dimerization of an initially generated formyl or oxymethylene complex, [(Me4TACD)Ca(OCH)]+.

Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code.

Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here, we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron-donating Nδ-methyl histidine (Me-His) with little effect on the enzyme structure. The rate of formation (k1) and the reactivity (k2) of compound I are unaffected by ligand substitution. In contrast, proton-coupled electron transfer to compound II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron donation and compound II reactivity, which can be explained by weaker electron donation from the Me-His ligand (“the push”) affording an electron-deficient ferryl oxygen with reduced proton affinity (“the pull”). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation designed to increase “the pull” by removing a hydrogen bond to the ferryl oxygen. Analogous substitutions in ascorbate peroxidase lead to similar activity trends to those observed in CcP, suggesting that a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how noncanonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.

Культурні реалії в англомовному художньому дискурсі та особливості їх перекладу

The article analyzes cultural realias in English language artistic discourse and peculiarities of their translation. Some of the manifestations of cultural labelling are realias that make up significant difficulties for translators. The concept of realia is clarified. Cultural-specific units include both realias and other designations of cultural-specific features, traditions and customs. Realia is considered as a unique object or phenomenon characteristic of one ethnic or linguistic community; a cultural equivalent of this object or phenomenon; a means of nominating the corresponding cultural equivalent in a language. Different scientists present many classifications of realias based on different principles. The most complete classification of realities was developed by S. Vlakhov and S. Florin, who propose to classify realities using their subject, local, temporary and translation divisions. The difficulties of translating realias are considered and relevant translation techniques are described. Strategies for the transfer of culturally specific units are distinguished. The problem of translating realias is one of the difficult issues of translation studies, which causes many differences and difficulties due to their form, features, word formation capabilities, as well as the borrowing mechanism and functioning as a borrowed word. The cultural content of the original text is determined by the presence of the realias that are typical for the bearers of the culture of the source language. Translation of realias requires a set of translation techniques that provide linguistic and cultural accommodation. The translator, who is responsible for the choice of translation strategy, largely determines the degree of reflection of cultural differences in translation. The translator must be able to recognize realias and make them understandable in the culture of the translation language. In order to correctly convey the realia, the translator needs to know exactly what this realia means and what background information is behind it. In addition, the peculiarities of translating cultural-specific units from English to Ukrainian are revealed. It is found out that the translation of realias in English-language artistic discourse implies using contextual analogue, hyperonymic substitution, calquing, transliteration and omission. The most frequent cases were contextual analog and hyperonymic substitution.

Theoretical Probe to the Mechanism of Pt-Catalyzed C-H Acylation Reaction: Possible Pathways for the Acylation Reaction of a Platinacycle.

Density functional theory (DFT) and nudged elastic band (NEB) theory have been used to study the possible pathways for the acylation of cycloplatinated complex A derived from 2-phenoxypyridine, which is conceived as the key step in the platinum-catalyzed acylation of 2-aryloxypyridines. Geometry optimization indicates that the previously proposed intermediate, an arenium ion species as a result of analogous aromatic substitution, is not an energy minimum, but rather cationic Pt-arene η2-complex E is obtained as a stable intermediate. NEB simulations suggest that the minimum energy pathway for the acylation reaction has energy barrier of 33.6 kcal/mol and consists of the following steps: (1) Nucleophilic substitution at acetyl chloride by the platinum of the reactant A forms five-coordinate Pt(IV) acylplatinum complex B with an energy barrier of 21.7 kcal/mol. (2) B undergoes 1,2-acyl migration from the platinum to the cyclometalated carbon through a three-membered platinacycle transition state to give Pt-arene η2-complex E with an energy barrier of 14.0 kcal/mol. (3) E undergoes ligand exchange with chloride to form neutral Pt-arene η2-complex F. (4) F undergoes ligand substitution with acetonitrile to give the product and the energy barrier is small (10.6 kcal/mol). The rate-determining step is the 1,2-acyl migration step. It is interesting to note that intermediate F was not included in the proposed mechanism but was identified by the NEB simulations. Five-coordinate Pt(IV) acylplatinum complex B undergoes barrierless ligand coordination with chloride to form neutral formal oxidative addition acylplatinum complex D; however, D is less stable than reactant A by 2.9 kcal/mol, which also implies that the isolation of an oxidative addition product Pt(IV) complex may be very challenging. The direct reductive elimination of D to form product P has a higher energy barrier (36.6 kcal/mol).

Unveiling the structural features of nonnative trimers of human superoxide dismutase 1

BackgroundHuman SOD1 contains a single tryptophan residue (W32) which has been identified as a site of oxidative modification and a potentiator of aggregation involving in familial amyotrophic lateral sclerosis (fALS). In situ substitution of a tryptophan analog, 2,6-diazatryptophan ((2,6-aza)Trp) with its unique water-catalyzed proton transfer property, into proteins exhibits extraordinary sensitivity in the detection of subtle water-associated structural changes with only a few micro-molar concentration of samples. MethodsA combination of size-exclusion chromatography and water-catalyzed fluorescent emission was utilized to probe the structural features of metastable SOD1 nonnative trimers, the potential neurotoxic species in the fALS. ResultsThe monomer of apo-A4V SOD1 exhibits variable conformations and the fastest trimeric formation rate compared to that of wild type and I113T. The trimeric A4V SOD1 exhibits the least water molecules surrounding the W32, while I113T and the wild type appear to have more water molecules in the proximity of W32. A small molecule stabilizer, 5-fluorouridine, effects the structural conformation of SOD1 nonnative trimers. ConclusionsOur studies unveil new insights into water-associated structural changes of SOD1 nonnative trimers and demonstrate that in situ incorporation of (2,6-aza)Trp is a sensitive and powerful tool for probing subtle changes of water environments during protein aggregation. General significanceThe water-sensitive probe, (2,6-aza)Trp, demonstrates superior sensitivity for detecting modulation of water microsolvation, structural conformation during oligomer formation and 5FUrd binding to both wild type and mutant SOD1.

PP2C phosphatase Pic1 negatively regulates the phosphorylation status of Pti1b kinase, a regulator of flagellin-triggered immunity in tomato.

Plant immune responses, including the production of reactive oxygen species (ROS), are triggered when pattern recognition receptors (PRRs) become activated upon detection of microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases are key components of PRR-dependent signaling pathways. In tomato, two such kinases, Pti1a and Pti1b, are important positive regulators of the plant immune response. However, it is unknown how these kinases control plant immunity at the molecular level and how their activity is regulated. To investigate these issues, we used mass spectrometry to search for interactors of Pti1b in Nicotiana benthamiana leaves and identified a PP2C protein phosphatase, referred to as Pic1. An in vitro pull-down assay and in vivo split-luciferase complementation assay verified this interaction. Pti1b was found to autophosphorylate on threonine-233, and this phosphorylation was abolished in the presence of Pic1. An arginine-to-cysteine substitution at position 240 in the Arabidopsis MARIS kinase was previously reported to convert it into a constitutive-active form. The analogous substitution in Pti1b made it resistant to Pic1 phosphatase activity, although it still interacted with Pic1. Treatment of N. benthamiana leaves with the MAMP flg22 induced threonine phosphorylation of Pti1b. The expression of Pic1, but not a phosphatase-inactive variant of this protein, in N. benthamiana leaves greatly reduced ROS production in response to treatment with MAMPs flg22 or csp22. The results indicate that Pic1 acts as a negative regulator by dephosphorylating the Pti1b kinase, thereby interfering with its ability to activate plant immune responses.

Structural basis for α-bungarotoxin insensitivity of neuronal nicotinic acetylcholine receptors

The ten types of nicotinic acetylcholine receptor α-subunits show substantial sequence homology, yet some types confer high affinity for α-bungarotoxin, whereas others confer negligible affinity. Combining sequence alignments with structural data reveals three residues unique to α-toxin-refractory α-subunits that coalesce within the 3D structure of the α4β2 receptor and are predicted to fit between loops I and II of α-bungarotoxin. Mutating any one of these residues, Lys189, Ile196 or Lys153, to the α-toxin-permissive counterpart fails to confer α-bungarotoxin binding. However, mutating both Lys189 and Ile196 affords α-bungarotoxin binding with an apparent dissociation constant of 104 nM, while combining mutation of Lys153 reduces the dissociation constant to 22 nM. Analogous residue substitutions also confer high affinity α-bungarotoxin binding upon α-toxin-refractory α2 and α3 subunits. α4β2 receptors engineered to bind α-bungarotoxin exhibit slow rates of α-toxin association and dissociation, and competition by cholinergic ligands typical of muscle nicotinic receptors. Receptors engineered to bind α-bungarotoxin co-sediment with muscle nicotinic receptors on sucrose gradients, and mirror single channel signatures of their α-toxin-refractory counterparts. Thus the inability of α-bungarotoxin to bind to neuronal nicotinic receptors arises from three unique and interdependent residues that coalesce within the receptor's 3D structure.

2-Hydroxypropyl-β-cyclodextrin is the active component in a triple combination formulation for treatment of Niemann-Pick C1 disease

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPβCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) – comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPβCD/PEG – shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPβCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.

Three Mutations in the Stalk Region of Hemagglutinin Affect the pH of Fusion and Pathogenicity of H5N1 Influenza Virus

Previously, an attenuated variant Ku/at was obtained from the highly pathogenic avian influenza virus A/chicken/Kurgan/3/2005 (H5N1) by a reverse selection method aimed at increasing the virus resistance to a proteolytic cleavage and acidic pH values. In the Ku/at, 10 mutations in proteins PB2, PB1, HA, NA, and NS1 occurred. In comparison with the parental strain, the pH of the conformational transition of the viral glycoprotein hemagglutinin (HA) and virulence for mice and chickens have decreased in an attenuated variant. The purpose of this work is to clarify the role of three mutations in the stalk region of HA: Asp54Asn in HA1 and Val48Ile and Lys131Thr in HA2 (H3 HA numbering). To attain these ends, analogous substitutions were introduced into HA with a deleted polybasic cleavage site (important for pathogenicity) of the recombinant A/Vietnam/1203/04-PR8/CDC-RG (H5N1) virus, and so we created the VN3x-PR variant. Viruses VN3x-PR and Ku/at with the same three mutations, but different proteolytic cleavage sites in HA, as well as the corresponding initial viruses, were tested for pathogenicity in mice and in the erythrocyte hemolysis test. Compared with the parental strains, the virulence of their mutant variants in the case of intranasal infection of BALB/c mice decreased by 4–5 orders of magnitude, and the pH of the conformational transition of HA decreased from 5.70–5.80 to 5.25–5.30, which is typical for low pathogenic natural isolates. Thus, as a result of the study, the attenuating role of these three mutations in HA has been proved, a correlation was established between the pH value of the HA conformational transition and the virulence of H5N1 influenza viruses, and it was shown that the polybasic cleavage site of the H5 HA does not always determine high pathogenicity of the virus.

Influence of Crystal Structure and 3d Impurities on the Electronic Structure of the Topological Material Cd3As2

This paper presents a theoretical study of the effects of crystal structure and Mn and Co substitutions for Cd on the electronic structure of the topological material Cd3As2. We have carried out density functional theory calculations of the band structure and density of states in tetragonal and cubic Cd3As2, as well as in Cd3 –xMnxAs2 and Cd3 –xCoxAs2 crystals. The results indicate that the band structure of the cubic Cd3As2 phase differs significantly from that of a Dirac semimetal, characteristic of the tetragonal phase. It has also been shown that, after Co substitution for 1/24 of the Cd atoms, the structure of the density of 3d electron states is similar to that of the density of states in the magnetic semiconductor Cd3 –xMnxAs2, with a characteristic minimum at the Fermi energy. At the same time, in the case of analogous Mn substitution for Cd, the density of d-electron states has no such minimum.

Drug Resistance in Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is a global protozoan parasite infecting up to one-third of the world population. Pyrimethamine (PYR) and sulfadiazine (SDZ) are the most widely used drugs for treatment of toxoplasmosis; however, several failure cases have been recorded as well; suggesting the existence of drug resistant strains. This review aims to give a systematic and comprehensive understanding of drug resistance in T. gondii including mechanisms of resistance and sites of drug action in parasite. Analogous amino acid substitutions in the Toxoplasma enzyme were identified to confer PYR resistance. Moreover, resistance to clindamycin, spiramycin, and azithromycin is encoded in the rRNA genes of T. gondii. However, T. gondii SDZ resistance mechanism has not been proved yet. Recently there has been a slight increase in SDZ resistance. That is why the majority of studies were carried out using SDZ. Six strains resistant to SDZ were found in clinical cases between 2013 and 2017 which among Brazilian T. gondii isolates, TgCTBr11, Ck3, and Pg1 were identified in human toxoplasmosis, as well as in livestock intended for human consumption. In conclusion, recent experimental studies in clinical cases have clearly shown that drug resistance in Toxoplasma is ongoing. Thus, establishing a more effective therapeutic scheme in the treatment of toxoplasmosis is critically needed. The emergence of T. gondii strains resistant to current drugs, reviewed here, represents a concern not only for treatment failure but also for increased clinical severity in immunocompromised patients. To improve the therapeutic outcome in patients, a greater understanding of the exact mechanisms of drug resistance in T. gondii should be developed. Thus, monitoring the presence of resistant parasites, in food products, would seem a prudent public health program.

Thermodynamic characterization for the denatured state of bovine prion protein and the BSE Associated variant E211K

ABSTRACTPropagation of transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. The most common hereditary prion disease is a genetic form of Creutzfeldt-Jakob disease in humans, in which a mutation in the prion gene results in a glutamic acid to lysine substitution at position 200 (E200K) in PrP. In cattle, the analogous amino acid substitution is found at residue 211 (E211K) and has been associated with a case of bovine spongiform encephalopathy. Here, we have compared the secondary structure of E211K to that of wild type using circular dichroism and completed a thermodynamic analysis of the folding of recombinant wild type and E211K variants of the bovine prion protein. The secondary structure of the E211K variant was essentially indistinguishable from that of wild type. The thermodynamic stability of E211K substitution showed a slight destabilization relative to the wild type consistent with results reported for recombinant human prion protein and its mutant E200K. In addition, the E211K variant exhibits a similarly compact denatured state to that of wild type based upon similar m-value and change in heat capacity of unfolding for the proteins. Together these results indicate that residual structure in the denatured state of bPrP is present in both the wild type protein and BSE associated variant E211K. Given this observation, as well as folding similarities reported for other disease associated variants of PrP it is worth consideration that functional aspects of PrP conformation may play a role in the misfolding process.

Cultivation and genomics of the first freshwater SAR11 (LD12) isolate

Evolutionary transitions between fresh and salt water happen infrequently among bacterioplankton. Within the ubiquitous and highly abundant heterotrophic Alphaproteobacteria order Pelagibacterales (SAR11), most members live in marine habitats, but the LD12 subclade has evolved as a unique freshwater lineage. LD12 cells occur as some of the most dominant freshwater bacterioplankton, yet this group has remained elusive to cultivation, hampering a more thorough understanding of its biology. Here, we report the first successful isolation of an LD12 representative, strain LSUCC0530, using high-throughput dilution-to-extinction cultivation methods, and its complete genome sequence. Growth experiments corroborate ecological data suggesting active populations of LD12 in brackish water up to salinities of ~5. LSUCC0530 has the smallest closed genome thus far reported for a SAR11 strain (1.16 Mbp). The genome affirms many previous metabolic predictions from cultivation-independent analyses, like a complete Embden–Meyerhof–Parnas glycolysis pathway, but also provides novel insights, such as the first isocitrate dehydrogenase in LD12, a likely homologous recombination of malate synthase from outside of the SAR11 clade, and analogous substitutions of ion transporters with others that occur throughout the rest of the SAR11 clade. Growth data support metagenomic recruitment results suggesting temperature-based ecotype diversification within LD12. Key gene losses for osmolyte uptake provide a succinct hypothesis for the evolutionary transition of LD12 from salt to freshwater. For strain LSUCC0530, we propose the provisional nomenclature Candidatus fonsibacter ubiquis.

Three Mutations in the Stalk Region of Hemagglutinin Affect the pH of Fusion and Pathogenicity of H5N1 Influenza Virus

Previously, an attenuated variant Ku/at was obtained from the highly pathogenic avian influenza virus A/chicken/Kurgan/3/2005 (H5N1) by a reverse selection method aimed at increasing the virus resistance to a proteolytic cleavage and acidic pH values. In the Ku/at, 10 mutations in proteins PB2, PB1, HA, NA, and NS1 occurred. In comparison with the parental strain, the pH of the conformational transition of the viral glycoprotein hemagglutinin (HA) and virulence for mice and chickens have decreased in an attenuated variant. The purpose of this work is to clarify the role of three mutations in the stalk region of HA: Asp54Asn in HA1 and Val48Ile and Lys131Thr in HA2 (H3 HA numbering). To attain these ends, analogous substitutions were introduced into HA with a deleted polybasic cleavage site (important for pathogenicity) of the recombinant A/Vietnam/1203/04-PR8/CDC-RG (H5N1) virus, and so we created the VN3x-PR variant. Viruses VN3x-PR and Ku/at with the same three mutations, but different proteolytic cleavage sites in HA, as well as the corresponding initial viruses, were tested for pathogenicity in mice and in the erythrocyte hemolysis test. Compared with the parental strains, the virulence of their mutant variants in the case of intranasal infection of BALB/c mice decreased by 4-5 orders of magnitude, and the pH of the conformational transition of HA decreased from 5.70-5.80 to 5.25-5.30, which is typical for low pathogenic natural isolates. Thus, as a result of the study, the attenuating role of these three mutations in HA has been proved, a correlation was established between the pH value of the HA conformational transition and the virulence of H5N1 influenza viruses, and it was shown that the polybasic cleavage site of the H5 HA does not always determine high pathogenicity of the virus.

Comparative analysis of inosine-substituted duplex DNA by circular dichroism and X-ray crystallography

Leveraging structural biology tools, we report the results of experiments seeking to determine if the different mechanical properties of DNA polymers with base analog substitutions can be attributed, at least in part, to induced changes from classical B-form DNA. The underlying hypothesis is that different inherent bending and twisting flexibilities may characterize non-canonical B-DNA, so that it is inappropriate to interpret mechanical changes caused by base analog substitution as resulting simply from ‘electrostatic’ or ‘base stacking’ influences without considering the larger context of altered helical geometry. Circular dichroism spectra of inosine-substituted oligonucleotides and longer base-substituted DNAs in solution indicated non-canonical helical conformations, with the degree of deviation from a standard B-form geometry depending on the number of I⋅C pairs. X-ray diffraction of a highly inosine-substituted DNA decamer crystal (eight I⋅C and two A⋅T pairs) revealed an A-tract-like conformation with a uniformly narrow minor groove, reduced helical rise, and the majority of sugars adopting a C1′-exo (southeastern) conformation. This contrasts with the standard B-DNA geometry with C2′-endo sugar puckers (south conformation). In contrast, the crystal structure of a decamer with only four I⋅C pairs has a geometry similar to that of the reference duplex with eight G⋅C and two A⋅T pairs. The unique crystal geometry of the inosine-rich duplex is noteworthy given its unusual CD signature in solution and the altered mechanical properties of some inosine-containing DNAs.