Minor Substitutions Research Articles

NAMBULITE AND NATRONAMBULITE OCCURRENCE IN THE MANGANESE BELT, BISTRIŢA MOUNTAINS,EASTERN CARPATHIANS, ROMANIA

Nambulite, (Li,Na)Mn4Si5O14(OH), and natronambulite, (Na,Li)(Mn,Ca)4Si5O14(OH), occur in many deposits of Manganese Belt (MnB) in Bistriţa Mountains, Eastern Carpathians, Romania. The nambulite and natronambulite form small bands which cut the old bands/lens of braunite-bixbyite-hausmannite ore. Nambulite has Li>Na and forms a series with natronambulite. The natronambulite has Na>Li and Mn>Ca. The X-ray analyses of MnB nambulite showed that it crystallizes in triclinic system, space group Pī(2). Its unit cell parameters are: a=7.5300 Å, b=11.7360 Å, c=6.7100 Å, Z=2, V=565.00 Å3, α=92.97°, β=95.23°, and γ=106.27°. The natronambulite has: a=7.6200 Å, b=11.7620 Å, c=6.7370 Å, Z=2, V=574.436 Å3, α=92.81°, β=94.55°, and γ= 106.87°. The cell parameters of natronambulite are very close to those of nambulite. The chemical compositions of nambulite and natronambulite of MnB deposits were determined by SEM and wet chemical analyses. Their Li2O and Na2O were determined by AAS and by wet chemical analyses. The wet chemical analyses of nambulite show variation for Li2O=1.05-1.34 wt.% and Na2O=1.28-0.72 wt.%. The natronambulite has Li2O< Na2O, and higher Na2O and CaO contents than nambulite. In natronambulite the Li2O content varies between 0.45-0.65 wt.% and that of Na2O between 1.10-5.96 wt.%. The main MnO compound for both minerals has variations between 38.20-46.01 wt.%, where Mn cations have minor substitutions with Mg, Ca, Ni and Zn. The mineral textural relations show that the oldest mineral of ore is braunite, closed associated with bixbyite/hausmannite. The braunite is cut/substituted by the new nambulite and natronambulite, which in their turn are substituted by leakeite and holmquistite, respectively. Both minerals are closely associated with aegirine and albite. A wide range of many minerals, some of them unusual and new for Romania, occur as veins through the nambulite/natronabulite associations: jacobsite-Q, namansilite, lithiomarsturite, serandite, hematophanite, fairfieldite, yoshimuraite, imandrite, and others. Also, many common minerals like magnetite, hematite, spessartine, albite, microcline, barium feldspars, barite, nickelian phlogopite/kinoshitalite, and others occur. The mineralogy, geochemistry, and tectonically setting of Mn deposits showed their hydrothermal submarine origin related to subduction processes. The source of Li was not from external supply.

Genome-based assessment of safety characteristics of Lacticaseibacillus paracasei NY1301 and genomic differences in closely related strains marketed as probiotics.

The probiotic attributes of Lacticaseibacillus paracasei NY1301 were comprehensively characterized, and a comparison between the closely related LcA (Actimel) and LcY (Yakult) probiotic strains was conducted using genomic tools. All strains exhibited high genetic similarity and likely shared a common ancestor; differences were primarily expressed as minor chromosomal re-arrangements, substitutions, insertions, and deletions. Compared with LcY, NY1301 exhibited 125 single-nucleotide polymorphisms. NY1301 lacked virulence factors, antibiotic resistance genes, and mutations associated with antibiotic resistance and had a 46-kbp prophage. This prophage is spontaneously induced at low levels and remains in a non-lytic state under standard culture conditions. The observed causal adaptive mutations were likely related to niche adaptation within the respective laboratory or manufacturing processes that occurred during the maintenance of the strains. However, the phenotypic effects of these genomic differences remain unclear. To validate the safety of NY1301, we conducted an open-label trial with healthy participants who consumed excessive amounts of NY1301 (3.0 × 1011 cfu) daily for 28 days. The results of this trial and those of other in vivo studies, coupled with the long history of human consumption without established risks to humans, provide strong evidence confirming the safety of NY1301.

Fine mapping of qBK1.2, a major QTL governing resistance to bakanae disease in rice.

Bakanae disease caused by Fusarium fujikuroi is an emerging disease of rice causing losses in all rice-growing regions around the world. A BC2F2 population was developed by backcrossing the recurrent parent Pusa Basmati 1121 (PB1121) with the recombinant inbred line RIL28, which harbors a major quantitative trait locus (QTL) governing resistance to bakanae, qBK1.2. MassARRAY-based single-nucleotide polymorphism (SNP) assays targeting the genomic region of qBK1.2 helped in fine mapping the QTL to a region of 130 kb between the SNP markers rs3164311 and rs3295562 using 24 recombinants. In-silico mining of the fine-mapped region identified 11 putative candidate genes with functions related to defense. The expression analysis identified two significantly differentially expressed genes, that is, LOC_Os01g06750 and LOC_Os01g06870, between the susceptible genotype PB1121 and the resistant genotypes Pusa1342 and R-NIL4. Furthermore, the SNPs identified in LOC_Os01g06750 produced minor substitutions of amino acids with no major effect on the resistance-related functional motifs. However, LOC_Os01g06870 had 21 amino acid substitutions, which led to the creation of the leucine-rich repeat (LRR) domain in the resistant genotype Pusa1342, thereby making it a potential candidate underlying the major bakanae-resistant QTL qBK1.2. The markers used in the fine mapping program are of immense utility in marker-assisted breeding for bakanae resistance in rice.

A Novel Isolate of Bean Common Mosaic Virus Isolated from Crownvetch (Securigera varia L. Lassen)

Bean common mosaic virus from the genus Potyvirus has a wide range of hosts and a very negative impact on cultivated crops from the genus Phaseolus. The risk of viral infection of economically important crops increases even if the carriers of the virus are related plant species growing on agroecological interfaces. Such plant species have emerged as new hosts for BCMV, usually harboring novel genetic variants of the virus. A novel genetic variant of BCMV was isolated from a symptomatic crownvetch plant, where the presence of this virus was confirmed via Western blot analysis and via amino acid identities in peptide fragments of CI, HC-pro, and CP proteins using the nanoLC-ESI-Q-TOF. The novel BCMV SVK isolate differed from the most genetically similar one in 0.91% of nucleotides and 1.55% of amino acids. The highest number of amino acid substitutions (8.8% of amino acids) was in the P1 protein, followed by CP (2.44% of amino acids). Minor substitutions were in Hc-pro, CI, and Nib proteins. The symptomatic crownvetch plant was confirmed as a new host and carrier of the novel BCMV isolate.

The Effect of Positive Charge Distribution on the Cryoprotective Activity of Dehydrins.

Dehydrins are intrinsically disordered proteins expressed ubiquitously throughout the plant kingdom in response to desiccation. Dehydrins have been found to have a cryoprotective effect on lactate dehydrogenase (LDH) in vitro, which is in large part influenced by their hydrodynamic radius rather than the order of the amino acids within the sequence (alternatively, this may be a sequence specific effect). However, it seems that a different mechanism may underpin the cryoprotection that they confer to the cold-labile yeast frataxin homolog-1 (Yfh1). Circular dichroism spectroscopy (CD) was used to assess the degree of helicity of Yfh1 at 1 °C, both alone and in the presence of several dehydrin constructs. Three constructs were compared to the wild type: YSK2-K→R (lysine residues substituted with arginine), YSK2-Neutral (locally neutralized charge), and YSK2-SpaceK (evenly distributed positive charge). The results show that sequence rearrangements and minor substitutions have little impact on the ability of the dehydrin to preserve LDH activity. However, when the positive charge of the dehydrin is locally neutralized or evenly distributed, the dehydrin becomes less efficient at promoting structure in Yfh1 at low temperatures. This suggests that a stabilizing, charge-based interaction occurs between dehydrins and Yfh1. Dehydrins are intrinsically disordered proteins, expressed by certain organisms to improve desiccation tolerance. These proteins are thought to serve many cellular roles, such as the stabilization of membranes, DNA, and proteins. However, the molecular mechanisms underlying the function of dehydrins are not well understood. Here, we examine the importance of positive charges in dehydrin sequences by making substitutions and comparing their effects in the cryoprotection of two different proteins.

Substrates and Inhibitors of the Organic Cation Transporter 3 and Comparison with OCT1 and OCT2.

Organic cation transporters (OCTs) 1, 2, and 3 facilitate cellular uptake of structurally diverse endogenous and exogenous substances. However, their substrate and inhibitor specificity are not fully understood. We performed a broad in vitro screening for OCT3 substrates and inhibitors, allowing us to compare the substrate spectra and to study the relationship between transport and inhibition of transport. Generally, substrates were smaller and more hydrophilic than OCT3 inhibitors. The best model-based predictor of transport was the positive charge, while the best predictor of inhibition was the aromatic ring count. OCT3 inhibition was well correlated between different model substrates. Substrates of OCT3 were mainly weak inhibitors, and the best inhibitors were not substrates. As tested with 264 substances, OCT3 transport had significantly more overlap with OCT2 than OCT1. Our data further substantiate that specificity of OCT transport varies with minor substitutions rather than with the general scaffolds of substrates.

Synthesis and characterisation of Fe-substituted Ni50Mn25Fe Ga25- single crystals—Development of the phase transformations with Fe content

Despite promising initial properties, the industrial applicability of magnetic shape memory alloys of the Ni-Mn-Ga family is distinctly limited by the temperature range it can operate in. Here we investigate the effects of minor substitutions of Fe for Ga on the thermodynamic, structural, and magnetic properties of Ni50Mn25FexGa25-x (3.0 < x < 7.5). Single crystals are essential for most intended applications, the studied compositions were prepared in single crystalline form. With increasing iron content, the melting point of the alloy decreases slightly from 1407(2) to 1394(2) K, while the structural B2’→L21 ordering transition increases in temperature from 1066(2) to 1082(4) K. Magnetisation measurements find that both the Curie temperature and the martensitic transformation temperature increase approximately linearly with Fe concentration, with Curie temperature increasing from 398(1) to 427(2) K, and martensitic transformation temperature increasing from 274(1) to 372(1) K when going from x = 3.0–7.5. The martensitic transformations above room temperature were confirmed by temperature-dependent single-crystal X-ray diffraction. These observations are compared with polycrystalline materials of the same series and support results of the previous studies. The shrinking gap between Curie and martensitic transformation temperatures, as well as the reduction in the low temperature saturated moment from 3.52(2) to 2.78(3) µB/f.u. make this substitution less industrially attractive but provide essential structural and property evolution details needed to improve and tune theoretical modelling of this important material family.

Spectroscopic investigation of the electronic and excited state properties of para-substituted tetraphenyl porphyrins and their electrochemically generated ions

Porphyrins play pivotal roles in many crucial biological processes including photosynthesis. However, there is still a knowledge gap in understanding electronic and excited state implications associated with functionalization of the porphyrin ring system. These effects can have electrochemical and spectroscopic signatures that reveal the complex nature of these somewhat minor substitutions, beyond simple inductive or electronic effect correlations. To obtain a deeper insight into the influences of porphyrin functionalization, four free-base, meso-substituted porphyrins: tetraphenyl porphyrin (TPP), tetra(4-hydroxyphenyl) porphyrin (THPP), tetra(4-carboxyphenyl) porphyrin (TCPP), and tetra(4-nitrophenyl) porphyrin (TNPP), were synthesized, characterized, and investigated. The influence of various substituents, (-hydroxy,-carboxy, and -nitro) in the para position of the meso-substituted phenyl moieties were evaluated by spectroelectrochemical techniques (absorption and fluorescence), femtosecond transient absorption spectroscopy, cyclic and differential pulse voltammetry, ultraviolet photoelectron spectroscopy (UPS), and time-dependent density functional theory (TD-DFT). Spectral features were evaluated for the neutral porphyrins and differences observed among the various porphyrins were further explained using rendered frontier molecular orbitals pertaining to the relevant transitions. Electrochemically generated anionic and cationic porphyrin species indicate similar absorbance spectroscopic signatures attributed to a red-shift in the Soret band. Emissive behavior reveals the emergence of one new fluorescence decay pathway for the ionic porphyrin, distinct from the neutral macrocycle. Femtosecond transient absorption spectroscopy analysis provided further analysis of the implications on the excited-state as a function of the para substituent of the free-base meso-substituted tetraphenyl porphyrins. Herein, we provide an in-depth and comprehensive analysis of the electronic and excited state effects associated with systematically varying the induced dipole at the methine bridge of the free-base porphyrin macrocycle and the spectroscopic signatures related to the neutral, anionic, and cationic species of these porphyrins.

A new emerald occurrence from Kruta Balka, Western Peri-Azovian region, Ukraine: Implications for understanding the crystal chemistry of emerald

AbstractWe investigated emerald, the bright-green gem variety of beryl, from a new locality at Kruta Balka, Ukraine, and compare its chemical characteristics with those of emeralds from selected occurrences worldwide (Austria, Australia, Colombia, South Africa, Russia) to clarify the types and amounts of substitutions as well as the factors controlling such substitutions. For selected crystals, Be and Li were determined by secondary ion mass spectrometry, which showed that the generally assumed value of 3 Be atoms per formula unit (apfu) is valid; only some samples such as the emerald from Kruta Balka deviate from this value (2.944 Be apfu). An important substitution in emerald (expressed as an exchange vector with the additive component Al2Be3Si6O18) is (Mg,Fe2+)NaAl–1☐–1, leading to a hypothetical end-member NaAl(Mg,Fe2+)[Be3Si6O18] called femag-beryl with Na occupying a vacancy position (☐) in the structural channels of beryl. Based on both our results and data from the literature, emeralds worldwide can be characterized based on the amount of femag-substitution. Other minor substitutions in Li-bearing emerald include the exchange vectors LiNa2Al–1☐–2 and LiNaBe–1☐–1, where the former is unique to the Kruta Balka emeralds. Rarely, some Li can also be situated at a channel site, based on stoichiometric considerations. Both Cr- and V-distribution can be very heterogeneous in individual crystals, as shown in the samples from Kruta Balka, Madagascar, and Zimbabwe. Nevertheless, taking average values available for emerald occurrences, the Cr/(Cr+V) ratio (Cr#) in combination with the Mg/(Mg+Fe) ratio (Mg#) and the amount of femag-substitution allows emerald occurrences to be characterized. The “ultramafic” schist-type emeralds with high Cr# and Mg# come from occur-rences where the Fe-Mg-Cr-V component is controlled by the presence of ultramafic meta-igneous rocks. Emeralds with highly variable Mg# come from “sedimentary” localities, where the Fe-Mg-Cr-V component is controlled by metamorphosed sediments such as black shales and carbonates. A “transitional” group has both metasediments and ultramafic rocks as country rocks. Most “ultramafic” schist type occurrences are characterized by a high amount of femag-component, whereas those from the “sedimentary” and “transitional” groups have low femag contents. Growth conditions derived from the zoning pattern—combined replacement, sector, and oscillatory zoning—in the Kruta Balka emeralds indicate disequilibrium growth from a fluid along with late-stage Na-infiltration. Inclusions in Kruta Balka emeralds (zircon with up to 11 wt% Hf, tourmaline, albite, Sc-bearing apatite) point to a pegmatitic origin.

Comparative analyses of phytochelatin synthase (PCS) genes in higher plants

Plants employ various defence strategies to ameliorate the effects of heavy metal exposures, leading to re-establishment of metal homeostasis. One of the strategies includes the biosynthesis of main heavy metal detoxifying peptides phytochelatins (PCs) by phytochelatin synthase (PCS). In the present study, 14 PCS homologues were identified in the genomes of 10 selected plants. The size of these PCSs was 452–545 amino acid residues, with characteristic phytochelatin and phytochelatin_C domains. The N-terminal site of the proteins is highly conserved, whereas the C-terminal site is less conserved. Further, the present study also identified fully conserved Cys residues involved in heavy metal binding reported earlier. In addition, other preserved cysteines, with minor substitutions Cys(C)→Ser(S) or Tyr(Y) or Trp(W), were also identified in the PCS sequences that might be associated with metal binding. The reported catalytic triad residues from Arabidopsis, Cys56, His162 and Asp180, are all conserved at the respective sites of PCSs. A clear monocot/dicot separation was revealed by phylogenetic analysis and was further corroborated by the exon–intron organisations of the PCS genes. Moreover, gene ontology terms, co-expression network, cis-regulatory motif and miRNA analyses indicated that the complex as well as dynamic regulation of PCSs has significant involvement in different metabolic pathways associated with signalling, defence, stress and phytohormone, in addition to metal detoxification. Moreover, variations in protein structure are suggested to confer the functional divergence in PCS proteins.

MicroRNA MIMIC binding sites: Minor flanking nucleotide alterations can strongly impact MIMIC silencing efficacy in Arabidopsis.

In plants, microRNA (miRNA) target MIMICs (MIMs) have been widely used to inhibit miRNA function. They are based on the Arabidopsis INSENSITIVE TO PHOSPATE STARVATION 1 (IPS1) gene that corresponds to a non‐coding RNA containing a miR399 binding site that can be modified to sequester and inhibit any miRNA of interest. However, the efficacy of miRNA inhibition of these different MIMs can vary greatly. Using MIMs that have strong efficacy (MIM159) and poor efficacy (MIM165), we investigate the underlying cause of this variation. Firstly, sequence alignments of IPS1 homologs from the Brassicaceae identified a highly conserved sequence immediately downstream of the miRNA binding site. Mutating this sequence in the context of the MIM159 attenuates its strong efficacy. This conserved flanking region contains a predicted stem‐loop structure that is also predicted to be present in most modified MIMs that appear to have a strong efficacy, but not in MIM165 that has a poor efficacy. Restoring this predicted stem‐loop in MIM165 via mutation of only three or five nucleotides within the conserved flanking region resulted in MIM165 variants that have very strong efficacies of miRNA inhibition. However, specifically mutating this predicted stem‐loop in the MIM159 context failed to significantly reduce efficacy, and additional mutations to restore this predicted stem‐loop weakened efficacy further. Although this shows there is no simple correlation between this predicted stem‐loop and efficacy, these results add to the growing evidence that the sequence context of miRNA binding sites is important, and that minor nucleotide substitutions to flanking sequences of miRNA binding sites can strongly enhance or attenuate the miRNA‐target interaction.

Thalidomide-type teratogenicity: structure-activity relationships for congeners.

Unravelling the molecular basis of thalidomide embryotoxicity, which is remarkably species-specific, is challenging in view of its low toxicity in the mature animal. Employing data derived solely from proven sensitive primate species or susceptible strains of rabbit, the structure-activity relationship of over 50 compounds which are, arguably, congeners of thalidomide has been reviewed. The molecular requirement for 'thalidomide-type' teratogenicity was highly structure dependent. Both the phthalimide and glutarimide groups were essential for embryopathic activity, although minor substitutions in either or both rings could be tolerated without a loss of toxicity. An α-linkage between the two cyclic structures was essential; a β-link resulted in a complete loss of embryopathic activity. Crucially, this α-configuration provided a centre of asymmetry enabling the existence of stereoisomers. The thalidomide molecule is not a static entity and under physiological conditions it undergoes a number of intra- and inter-molecular reactions. Besides irreversible hydrolysis, its keto-enol tautomerism, base-assisted proton transfer and glutarimide ring rotation lead to rapid interconversion of the thalidomide enantiomers. These enantiomers form equilibria between themselves and also between both homochiral and heterochiral dimers. It is proposed that the more energetically favourable and stable heterochiral dimer of thalidomide is an active agent that possesses the structural features of the paired nucleotides of the double-stranded DNA. Its capacity to enter into hydrogen bonding interactions affects DNA expression in a chaotic manner without causing permanent mutations. This disruption may well be concentrated at nucleotide sites known to be involved in specific promoter regions of the genome.

Chemical and boron isotopic compositions of tourmaline in the Longtoushan hydrothermal gold deposit, Guangxi, China: implications for gold mineralization

The Longtoushan hydrothermal gold deposit is located in the southwestern region of the Dayaoshan Uplift. Tourmaline is widespread in the Longtoushan gold deposit and is mainly distributed in the rhyolite porphyry and associated cryptoexplosive breccia. The spatial distribution of tourmaline enrichment is similar to that of the gold orebody. Feldspar has been largely replaced by tourmaline in the rhyolite porphyry and cryptoexplosive breccia. Electron microprobe analysis revealed that tourmalines in the Longtoushan deposit belong mainly to the alkali group and partly to the X-vacant group; they mostly fell in the schorl-dravite series field. Two distinct sets of dominant substitutions were observed: MgFe−1 and Al□(NaR2+)−1, where R = Fe, Mg. In addition, minor substitutions include (CaMg)(NaAl)−1 and FeAl−1. The calculated δ11B value for the mineralizing fluids ranged from − 12.8 to − 9.7‰, which is typical of S-type granites, and boron-enriched fluids predominantly derived from rhyolitic melt. Part of the tourmaline from the rhyolite porphyry crystallized during the magmatic-hydrothermal stage, whereas most tourmalines from the deposit formed in the post-magmatic hydrothermal stage. The tourmalines were deposited from a relatively reduced and acidic fluid system, and the gold predominantly precipitated during the post-magmatic hydrothermal stage in the Longtoushan deposit.

Raman spectroscopy of shocked enstatite‐rich meteorites

AbstractWe present Raman patterns of enstatite in different classes of enstatite‐rich chondrites and achondrites of various shock levels as previously reported from petrographic observations and X‐ray diffraction analyses. Thin sections or mineral separates of four enstatite chondrites (LaPaz Icefield [LAP] 02225, MacAlpine Hills [MAC] 02837, Pecora Escarpment [PCA] 91020, and Itqiy), three aubrites (Larkman Nunatak [LAR] 04316, Khor Temiki, and Allan Hills [ALH] 84008), and a ureilite (Sayh al Uhaymir [SaU] 559) were examined by laser Raman spectroscopy. We find that the frequencies of fundamental Raman peaks of enstatites from the chondrites and aubrites deviate by ≤2 cm−1 from the values for unshocked enstatite. This small difference implies a negligible effect of shock metamorphism on peak positions. Significant differences (&lt;6 cm−1) for peak positions are found for the pyroxenes of SaU 559 and may be attributed to minor substitution of Fe and Ca for Mg. Linear regressions of peak widths of enstatite chondrites against their established shock stages show a strong positive correlation for each mode (r2 &gt; 0.94). From this linear relationship, the 343 and 1014 cm−1 peaks of the aubrites coincide with S4 determined from petrography. For Itqiy, we find S4–5, while the shock levels of SaU 559 exceed the petrologic scheme (S1–6), suggesting that the ureilite might have sustained multiple shock events or have been deformed in a high‐pressure environment. Alternatively, for Itqiy (peak 343 cm−1) and SaU 559 (all peaks) enstatites, minor substitutions of Fe and Ca for Mg may have further broadened their peak widths.

The treatment outcomes of antiretroviral substitutions in routine clinical settings in Asia; data from the TREAT Asia HIV Observational Database (TAHOD).

IntroductionAlthough substitutions of antiretroviral regimen are generally safe, most data on substitutions are based on results from clinical trials. The objective of this study was to evaluate the treatment outcomes of substituting antiretroviral regimen in virologically suppressed HIV‐infected patients in non‐clinical trial settings in Asian countries.MethodsThe study population consisted of HIV‐infected patients enrolled in the TREAT Asia HIV Observational Database (TAHOD). Individuals were included in this analysis if they started combination antiretroviral treatment (cART) after 2002, were being treated at a centre that documented a median rate of viral load monitoring ≥0.8 tests/patient/year among TAHOD enrolees, and experienced a minor or major treatment substitution while on virally suppressive cART. The primary endpoint to evaluate outcomes was clinical or virological failure (VF), followed by an ART class change. Clinical failure was defined as death or an AIDS diagnosis. VF was defined as confirmed viral load measurements ≥400 copies/mL followed by an ART class change within six months. Minor regimen substitutions were defined as within‐class changes and major regimen substitutions were defined as changes to a drug class. The patterns of substitutions and rate of clinical or VF after substitutions were analyzed.ResultsOf 3994 adults who started ART after 2002, 3119 (78.1%) had at least one period of virological suppression. Among these, 1170 (37.5%) underwent a minor regimen substitution, and 296 (9.5%) underwent a major regimen substitution during suppression. The rates of clinical or VF were 1.48/100 person years (95% CI 1.14 to 1.91) in the minor substitution group, 2.85/100 person years (95% CI 1.88 to 4.33) in the major substitution group and 2.53/100 person years (95% CI 2.20 to 2.92) among patients that did not undergo a treatment substitution.ConclusionsThe rate of clinical or VF was low in both major and minor substitution groups, showing that regimen substitution is generally effective in non‐clinical trial settings in Asian countries.

HIV Type 1 Integrase Natural Polymorphisms in Viral Variants Circulating in FSU Countries.

Natural variability of integrase (IN) across HIV-1 variants may influence the emergence of resistant viruses. The most apparent explanation of this fact is the IN polymorphism and the associated differences in codon usage, which in turn, influence the probability and the terms of DRMs acquisition. Possible mechanisms by which polymorphisms affect DRMs emergence remain disputed and should still be clarified because these substitutions may be associated with a reduced activity of some INSTIs and may impact on ART regimen choice depending of HIV-1 subtype. The aim of this work was to assess the prevalence of naturally occurring polymorphisms within the HIV-1 integrase gene, which might influence the susceptibility to INSTIs, among the patients from Russia and former USSR countries, according to HIV-1 subtypes. A study involved 506 HIV-1 IN sequences of INSTI-naive patients from Russia, Ukraine, Armenia, Kyrgyzstan, Kazakhstan, Uzbekistan, Belarus, and Georgia. Among them, 194 sequences were newly obtained in this study and 312 were downloaded from Los-Alamos database. The proviral DNA was sequenced using an in-house PCR protocol designed on the basis of a well-conserved integrase region in order to detect all HIV-1 variants. The phylogenetic analyses based on IN population sequencing found subtype A6 being the most prevalent (259) (51.2%) in the collection studied, followed by subtype G (36) (7.1%), AGrecombinants (148) (29.3%), subtype B (50) (9.9%), and CRF03_AB (5) (1.0%). The major INSTI resistance-associated mutations (DRMs) were found only in two A6 samples. The prevalence of minor/ accessory substitutions depended on HIV-1 variants, while the most notable findings were L74I in subtype A6 (93.1%) and E157Q in subtype B (44.0%). Most of minor DRMs and polymorphic substitutions were concentrated in the central catalytic domain of the IN molecule. Both the DDE triad and HHCC zinc binding motifs were fully conserved. The results of the study suggest a very low risk of initiating INSTI-based therapy in patients with pre-existing polymorphic mutations in Russia and FSU countries. The therapy response in dominating HIV-1 genetic variants might be further studied in the future for a better understanding of their effect on INSTI susceptibility. The INSTI TDR is absent for the moment, but the risk may increase with expanded use of INSTIs, indicating the need for ongoing surveillance.

Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava.

CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (Manihot esculenta), the Phytoene desaturase (MePDS) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium-mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted MePDS site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 5′ and or downstream of the 3′ targeted MePDS region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of MePDS described here generates visually detectable mutated events in a relatively short time frame of 6–8 weeks, and does not require sequencing to confirm editing at the target. It therefore provides a valuable platform to facilitate rapid assessment and optimization of CRISPR/Cas9 and other genome-editing technologies in cassava.

The Safety of Substitution of Antiretroviral Regimen in Non-Clinical Trial Settings in Asian Countries

BackgroundAlthough substitutions of antiretroviral regimen are generally safe, most data on substitutions are based on results from clinical trials. The objective of this study was to evaluate the safety of substituting antiretroviral regimen in virologically suppressed HIV-infected patients in non-clinical trial settings in Asian countries.MethodsHIV-infected patients enrolled in the TREAT Asia HIV Observational Database (TAHOD) were included in this analysis if they started combination antiretroviral therapy (cART) after 2002, were being treated at a center that documented a median rate of viral load (VL) monitoring ≥ 1 tests/patient/year, and experienced a minor or major treatment substitution while on virally suppressive cART (VL < 200 copies/mL). Minor regimen substitutions were defined as within-class changes and major regimen substitutions were defined as changes to a drug class. Virologic failure was defined as having had two viral load measurements > 400 copies/mL. The patterns of substitutions and rate of virologic failure after substitutions were analyzed.ResultsOf 3,994 adults who started ART after 2002, 3,119 (78.1%) had at least one period of virological suppression. Among these, 1,170 (37.5%) underwent a minor regimen substitution, and 296 (9.5%) underwent a major regimen substitution during suppression. The rates of virological failure were 1.48/100person years (95% CI 1.14–1.91) in the minor substitution group and 2.85/100person years (95% CI 1.88–4.33) in the major substitution group, and 2.53/100person years (95% CI 2.20–2.92) among patients that did not undergo a treatment substitution.ConclusionThe rate of virological failure was relatively low in both major and minor substitution groups, showing that regimen substitution is generally safe in non-clinical trial settings in Asian countries.Disclosures All authors: No reported disclosures.

Modulation of anti-endotoxin property of Temporin L by minor amino acid substitution in identified phenylalanine zipper sequence.

A 13-residue frog antimicrobial peptide Temporin L (TempL) possesses versatile antimicrobial activities and is considered a lead molecule for the development of new antimicrobial agents. To find out the amino acid sequences that influence the anti-microbial property of TempL, a phenylalanine zipper-like sequence was identified in it which was not reported earlier. Several alanine-substituted analogs and a scrambled peptide having the same composition of TempL were designed for evaluating the role of this motif. To investigate whether leucine residues instead of phenylalanine residues at 'a' and/or 'd' position(s) of the heptad repeat sequence could alter its antimicrobial property, several TempL analogs were synthesized after replacing these phenylalanine residues with leucine residues. Replacing phenylalanine residues with alanine residues in the phenylalanine zipper sequence significantly compromised the anti-endotoxin property of TempL. This is evident from the higher production of tumor necrosis factor-α and interleukin-6 in lipopolysaccharide (LPS)-stimulated rat bone-marrow-derived macrophage cells in the presence of its alanine-substituted analogs than TempL itself. However, replacement of these phenylalanine residues with leucine residues significantly augmented anti-endotoxin property of TempL. A single alanine-substituted TempL analog (F8A-TempL) showed significantly reduced cytotoxicity but retained the antibacterial activity of TempL, while the two single leucine-substituted analogs (F5L-TempL and F8L-TempL), although exhibiting lower cytotoxicity, were able to retain the antibacterial activity of the parent peptide. The results demonstrate how minor amino acid substitutions in the identified phenylalanine zipper sequence in TempL could yield analogs with better antibacterial and/or anti-endotoxin properties with their plausible mechanism of action.

Enthalpy-Entropy Compensation in the Binding of Modulators at Ionotropic Glutamate Receptor GluA2

The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer’s disease. The modulators bind in a cleft formed by the interface of two neighboring ligand binding domains and act by stabilizing the agonist-bound open-channel conformation. The driving forces behind the binding of these modulators can be significantly altered with only minor substitutions to the parent molecules. In this study, we show that changing the 7-fluorine substituent of modulators BPAM97 (2) and BPAM344 (3) into a hydroxyl group (BPAM557 (4) and BPAM521 (5), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from −4.9 (2) and −7.5 (3) to −6.2 (4) and −14.5 (5), but also a less favorable binding entropy (−TΔS, kcal/mol) from −2.3 (2) and −1.3 (3) to −0.5 (4) and 4.8 (5). Thus, the dissociation constants (Kd, μM) of 4 (11.2) and 5 (0.16) are similar to those of 2 (5.6) and 3 (0.35). Functionally, 4 and 5 potentiated responses of 10 μM L-glutamate at homomeric rat GluA2(Q)i receptors with EC50 values of 67.3 and 2.45 μM, respectively. The binding mode of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5. The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser-497 to the hydroxyl group of 5, whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5. In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation of the underlying structural mechanism.