Isoliquiritigenin: a potential drug candidate for the management of erectile dysfunction.

OBJECTIVE: This study aimed to assess the erectogenic properties of isoliquiritigenin taking sildenafil (SDF) as the standard. METHODS: The binding affinity of isoliquiritigenin (ISL) with the erectile marker proteins (endothelial nitric oxide synthase [eNOS] and enzyme phosphodiesterase type 5 [PDE5]) was investigated using Autodock Vina, which was validated using molecular dynamics simulation. Furthermore, the effect of ISL on the eNOS and PDE5 messenger ribonucleic acid (mRNA) expression and the sexual behavior of mice was investigated, along with the assessment of the pharmacokinetics of ISL. KEY FINDINGS: The results revealed that the binding affinity of ISL-eNOS/PDE5 and SDF-eNOS/PDE5 was in the range of -7.5 to -8.6 kcal/mol. The ISL-eNOS/PDE5 complexes remained stable throughout the 100 ns simulation period. Root mean square deviation, Rg, SASA, hydrogen, and hydrophobic interactions were similar between ISL-eNOS/PDE5 and SDF-eNOS/PDE5. Analysis of mRNA expressions in paroxetine (PRX)-induced ED mice showed that the co-administration of PRX with ISL reduced PDE5 and increased eNOS mRNA expression, similar to the co-administered group (PRX+SDF). The sexual behavior study revealed that the results of PRX+ISL were better than those of the PRX+SDF group. Pharmacokinetic evaluation further demonstrated that ISL possesses drug-like properties. CONCLUSIONS: The results showed that ISL is equally potent as SDF in terms of binding affinity, specific pharmacological properties, and modulating sexual behavior.

Assessing the performance of farm soil-based and hybrid biofilters for methane abatement.

Mitigating methane (CH4) emissions using methanotrophs (methane-oxidizing bacteria, MOB), is a simple, energy efficient and cheap technology. The abundance and distribution of MOB in the environmental samples is critical for efficient removal of emitted CH4 from any source. This study evaluated the performance of farm soils without and with cheap, easily accessible bulking materials as sustainable hybrid biofilter media. Soil-only biofilters removed up to 865 ± 19 g CH4 m-3 d-1 with well-drained organic carbon-rich soils compared with 264 ± 14 g CH4 m-3 d-1 for poorly drained soil. The removal efficiency decreased with increasing flow rate (0.16→0.24 L min-1) and subsequent priming could not return soil biofilters to their previous removal rate.Hybrid biofilters using organic, carbon-rich soils and compost removed up to 2698 g CH4 m-3 d-1 (flow rate 0.35 L min-1). Increasing CH4 flow rates also reduced their efficiency, but the hybrid biofilters with compost quickly regained most of their efficiency and removed up to 2262 g CH4 m-3 d-1 (flow rate 0.3 L min-1) after remixing of biofilter media. These results show that hybrid biofilters removed higher CH4 than soil-only biofilters and were also more resilient. The MOB gene abundance results complement the CH4 removal capacity of both soil-only and hybrid biofilter materials used. The more aerobic, carbon-rich soils had more abundant MOB than the poorly drained soil. The most porous hybrid biofilter with compost and more available nutrients to sustain bacterial growth and activity had the highest MOB abundance and removed the most CH4.

Structure and Function of a Dehydrating Condensation Domain in Nonribosomal Peptide Biosynthesis.

Dehydroamino acids are important structural motifs and biosynthetic intermediates for natural products. Many bioactive natural products of nonribosomal origin contain dehydroamino acids; however, the biosynthesis of dehydroamino acids in most nonribosomal peptides is not well understood. Here, we provide biochemical and bioinformatic evidence in support of the role of a unique class of condensation domains in dehydration (CmodAA). We also obtain the crystal structure of a CmodAA domain, which is part of the nonribosomal peptide synthetase AmbE in the biosynthesis of the antibiotic methoxyvinylglycine. Biochemical analysis reveals that AmbE-CmodAA modifies a peptide substrate that is attached to the donor carrier protein. Mutational studies of AmbE-CmodAA identify several key residues for activity, including four residues that are mostly conserved in the CmodAA subfamily. Alanine mutation of these conserved residues either significantly increases or decreases AmbE activity. AmbE exhibits a dimeric conformation, which is uncommon and could enable transfer of an intermediate between different protomers. Our discovery highlights a central dehydrating function for CmodAA domains that unifies dehydroamino acid biosynthesis in diverse nonribosomal peptide pathways. Our work also begins to shed light on the mechanism of CmodAA domains. Understanding CmodAA domain function may facilitate identification of new natural products that contain dehydroamino acids and enable engineering of dehydroamino acids into nonribosomal peptides.

KasQ an Epimerase Primes the Biosynthesis of Aminoglycoside Antibiotic Kasugamycin and KasF/H Acetyltransferases Inactivate Its Activity.

Kasugamycin (KSM), an aminoglycoside antibiotic, is composed of three chemical moieties: D-chiro-inositol, kasugamine and glycine imine. Despite being discovered more than 50 years ago, the biosynthetic pathway of KSM remains an unresolved puzzle. Here we report a structural and functional analysis for an epimerase, KasQ, that primes KSM biosynthesis rather than the previously proposed KasF/H, which instead acts as an acetyltransferase, inactivating KSM. Our biochemical and biophysical analysis determined that KasQ converts UDP-GlcNAc to UDP-ManNAc as the initial step in the biosynthetic pathway. The isotope-feeding study further confirmed that 13C, 15N-glucosamine/UDP-GlcNH2 rather than glucose/UDP-Glc serves as the direct precursor for the formation of KSM. Both KasF and KasH were proposed, respectively, converting UDP-GlcNH2 and KSM to UDP-GlcNAc and 2-N'-acetyl KSM. Experimentally, KasF is unable to do so; both KasF and KasH are instead KSM-modifying enzymes, while the latter is more specific and reactive than the former in terms of the extent of resistance. The information gained here lays the foundation for mapping out the complete KSM biosynthetic pathway.

Persistent susceptibility of Aedes aegypti to eugenol.

Botanical insecticides are preferred for their environment and user-friendly nature. Eugenol is a plant-based monoterpene having multifarious biocidal activities. To understand whether eugenol would persistently work against Aedes aegypti, we performed larvicidal bioassays on thirty successive generations and determined median lethal concentration (LC50) on each generation. Results showed no apparent differences between LC50 at F0 (63.48 ppm) and F30 (64.50 ppm) indicating no alteration of susceptibility toward eugenol. To analyze, if eugenol has any effect on metabolic detoxification-associated enzymes, we measured esterases (alpha and beta), cytochrome P450, and GST activities from the survived larvae exposed to LC50 concentration from F0-F30. Results revealed a decrease of esterases, GST, and cytochrome P450 activities at the initial 4-8 generations and then a gradual increase as the generations progressed. GST activity remained significantly below the control groups. Synergists (TPP, DEM, and PBO) were applied along with eugenol at F30 and LC50 concentration, and the said enzyme activities were recorded. Results showed a noticeable decrease in LC50 and enzyme activities indicating effective inhibitions of the respective enzymes. Overall, present results inferred that eugenol would effectively work as a larvicide for a longer period in successive generations without initiating rapid resistance and therefore could be advocated for controlling A. aegypti.

Improving the accuracy of nitrous oxide emission factors estimated for hotspots within dairy-grazed farms.

Nitrous oxide (N2O) emissions from dairy-grazing pastures can be dominated by large emissions from small areas ('hotspots') frequently used by grazing dairy cattle (i.e., water troughs and gateways). N2O emissions from these hotspots are quantified by investigating whether N2O emissions and emission factors (% of applied N emitted as N2O, EF3) from potential hotspots are different from non-hotspots. To better characterise N2O emissions from hotspots and non-hotspots of farms to understand their contributions to national agricultural greenhouse gas inventory calculations, a series of measurements were conducted during winter and spring on two NZ typical dairy farms with contrasting soil drainage (poorly versus well drained). Before measurements were taken, the soils either received a cow urine application or remained untreated. The results showed that changes in water-filled pore space (WFPS) and mineral N around water troughs and gateways, due to additional stock movements and disproportionate excreta-N deposition during previous grazing events, affected both background and total N2O emissions. But there was little impact on EF3 values (calculated using IPCC guidelines) from deposited urine between hotspot and pasture areas. These results suggest the same EF3 values can be used for both to calculate emissions from urine deposited on grazed pastures. However, these results raise concerns about higher background emission in hotspots subtracted from measured emissions from urine-N deposition in calculating EF3 values and discounting the effects of disproportionate N inputs in intensive agriculture on increased background emissions (legacy effect). This IPCC inventory method does not account for the legacy effect of N loading prior to the measurements which may underestimate the emissions. Thus, an allowance for higher hotspot background emissions could be included in the Inventory to accurately estimate total emissions from agriculture.

ß-Hydroxylation of α-amino-ß-hydroxylbutanoyl-glycyluridine catalyzed by a nonheme hydroxylase ensures the maturation of caprazamycin.

Caprazamycin is a nucleoside antibiotic that inhibits phospho-N-acetylmuramyl-pentapeptide translocase (MraY). The biosynthesis of nucleoside antibiotics has been studied but is still far from completion. The present study characterized enzymes Cpz10, Cpz15, Cpz27, Mur17, Mur23 out of caprazamycin/muraymycin biosynthetic gene cluster, particularly the nonheme αKG-dependent enzyme Cpz10. Cpz15 is a ß-hydroxylase converting uridine mono-phosphate to uridine 5' aldehyde, then incorporating with threonine by Mur17 (Cpz14) to form 5'-C-glycyluridine. Cpz10 hydroxylates synthetic 11 to 12 in vitro. Major product 13 derived from mutant Δcpz10 is phosphorylated by Cpz27. ß-Hydroxylation of 11 by Cpz10 permits the maturation of caprazamycin, but decarboxylation of 11 by Mur23 oriented to muraymycin formation. Cpz10 recruits two iron atoms to activate dioxygen with regio-/stereo-specificity and commit electron/charge transfer, respectively. The chemo-physical interrogations should greatly advance our understanding of caprazamycin biosynthesis, which is conducive to pathway/protein engineering for developing more effective nucleoside antibiotics.

Gradual reduction of susceptibility and enhanced detoxifying enzyme activities of laboratory-reared Aedes aegypti under exposure of temephos for 28 generations.

Temephos, an organophosphate insecticide, is widely accepted for the control of Aedes aegypti, vector of infectious diseases such as dengue, chikungunya, yellow fever, and zika. However, there are claims that repeated and indiscriminate use of temephos has resulted in resistance development in exposed mosquito populations. The present study attempts to evaluate the continuous performance of temephos on the Ae. aegypti population, in laboratory conditions, in terms of toxicity and the effect on marker enzymes associated with metabolic resistance. Results of the toxicity bioassay showed that after the initial exposure, toxicity increased till F4 generation by 1.65 fold, and continuous exposure resulted in a 7.83 fold reduction in toxicity at F28 generation. Percent mortality result showed a marked reduction in mortality with the passage of generations while using the same series of concentrations, viz. 2 ppm, which was 100 % lethal at the initial nine generations, could kill only 22.66 % at F28. Resistance to organophosphates is mainly governed by metabolic detoxifying enzyme families of esterases, glutathione-s-transferase, and cytochrome P450. Analysis of these metabolic detoxifying enzymes showed an inverse trend to toxicity (i.e. toxicity increased in early generations as enzyme activity dropped and then dropped as enzyme activity increased). At the initial exposure, enzyme activity decreased in 2-4 generations, however, repeated exposure led to a significant increase in all the metabolic detoxifying enzymes. From the toxicity level as well as marker enzyme bioassay results, it can be inferred that mosquitoes showed increased detoxification in generational time with an increase in enzymes associated with metabolic detoxification. In conclusion, repeated application of temephos led to resistance development in Ae. aegypti which may be associated with the increase in metabolic detoxifying enzyme activities.

Management and implications of using nitrification inhibitors to reduce nitrous oxide emissions from urine patches on grazed pasture soils - A review.

Livestock urine patches are the main source of nitrous oxide (N2O) emissions in pastoral system, and nitrification inhibitors (NIs) have been widely investigated as a N2O mitigation strategy. This study reviews the current understanding of the effect of NIs use on N2O emissions from urine patches, including the factors that affect their efficacy, as well as the unintended consequences of NIs use. It brings together the fundamental aspects of targeted management of urine patches for reducing N2O emissions involving inhibitors. The available literature of 196 datasets indicates that dicyandiamide (DCD), 3,4-dimethylpyrazole phosphate (DMPP), and 2-chloro-6-(trichloromethyl) pyridine (nitrapyrin) reduced N2O emissions from urine patches by 44 ± 2%, 28 ± 38% and 28 ± 5%, (average ± s.e.), respectively. DCD also increased pasture dry matter and nitrogen (N) uptake by 13 ± 2% and 15 ± 3%, (average ± s.e.), respectively. The effect of DMPP and nitrapyrin on pasture dry matter and N uptake, assessed in only one study, was not significant. It also suggests that harmonizing the timing of inhibitor use with urine-N transformation increase the efficacy of NIs. No negative impacts on non-targeted soil and aquatic organisms have been reported with the recommended rate of DCD applied to urine and recommended applications of DMPP and nitrapyrin for treated mineral fertilisers and manures. However, there was evidence of the presence of small amounts of DCD residues in milk products as a result of its use on livestock grazed pasture. DMPP and nitrapyrin can also enter the food chain via grazing livestock. The study concludes that for the use of NIs in livestock grazed systems, research is needed to establish acceptable maximum residue level (MRL) of NIs in soil, plant, and animal products, and develop technologies that optimise physical mixing between NIs and urine patches.

Theoretical Study of Intermolecular Interactions between Critical Residues of Membrane Protein MraYAA and Promising Antibiotic Muraymycin D2.

Phospho-N-acetylmuramoyl-pentapeptide translocase (MraYAA) from Aquifex aeolicus is the binding target for the nucleotide antibiotic muraymycin D2 (MD2). MraYAA in the presence of the MD2 ligand has been crystallized and released, while the interactions between the ligand and active-site residues remain less quantitatively and qualitatively defined. We characterized theoretically the key residues involved in noncovalent interactions with MD2 in the MraYAA active site. We applied the quantum theory of atoms in molecules and natural bond orbital analyses based on the density functional theory method on the solved crystal structure of MraY with the MD2 to quantitatively estimate the intermolecular interactions. The obtained results revealed the presence of multiple hydrogen bonds in the investigated active site with strength ranging from van der Waals to covalent limits. Lys70, Asp193, Gly194, Asp196, Gly264, Ala321, Gln305, and His325 are key active-site residues interacting with MD2. Conventional and unconventional hydrogen bonds in addition with charge-dipole and dipole-dipole interactions contribute significantly to stabilize the MD2 binding to the MraYAA active site. It was also found that water molecules inside the active site have substantial effects on its structure stability through hydrogen-bonding interactions with MD2 and the interacting residues.

Chemoenzymatic Synthesis and Biological Evaluation for Bioactive Molecules Derived from Bacterial Benzoyl Coenzyme A Ligase and Plant Type III Polyketide Synthase.

Plant type III polyketide synthases produce diverse bioactive molecules with a great medicinal significance to human diseases. Here, we demonstrated versatility of a stilbene synthase (STS) from Pinus Sylvestris, which can accept various non-physiological substrates to form unnatural polyketide products. Three enzymes (4-coumarate CoA ligase, malonyl-CoA synthetase and engineered benzoate CoA ligase) along with synthetic chemistry was practiced to synthesize starter and extender substrates for STS. Of these, the crystal structures of benzoate CoA ligase (BadA) from Rhodopseudomonas palustris in an apo form or in complex with a 2-chloro-1,3-thiazole-5-carboxyl-AMP or 2-methylthiazole-5-carboxyl-AMP intermediate were determined at resolutions of 1.57 Å, 1.7 Å, and 2.13 Å, respectively, which reinforces its capacity in production of unusual CoA starters. STS exhibits broad substrate promiscuity effectively affording structurally diverse polyketide products. Seven novel products showed desired cytotoxicity against a panel of cancer cell lines (A549, HCT116, Cal27). With the treatment of two selected compounds, the cancer cells underwent cell apoptosis in a dose-dependent manner. The precursor-directed biosynthesis alongside structure-guided enzyme engineering greatly expands the pharmaceutical repertoire of lead compounds with promising/enhanced biological activities.

Insecticidal activities of Citrus aurantifolia essential oil against Aedes aegypti (Diptera: Culicidae).

BACKGROUND: In the recent time, global attention for the control of vectors has shifted from chemical insecticides to botanicals. In the present investigation, authors attempted to evaluate the efficacy of peel and leaf essential oil (EO) of Citrus aurantifolia against Aedes aegypti. RESULTS: The results revealed that both the oils possess more ovicidal activity (LC50 value of 5.26 ppm and 17.71 ppm for leaf and peel oil respectively at 72 h) than larvicidal activity. As larvicide, the essential oil from the peel of Citrus aurantifolia showed rapid effect with LC50 value of 128.81 ppm at 24 h which reduced to 106.77 ppm at 72 h while the leaf oil showed slow effect with LC50 value of 188.59 ppm, 107.37 ppm and 104.59 ppm at 24 h, 48 h and 72 h respectively. Again, the two essential oils did not show significant adulticidal activity. GC-MS analysis of both the oils recorded presence of different compounds. As a major constituent compound of the leaf EO of Citrus aurantifolia, citral was tested for their ovicidal, larvicidal and adulticidal activities against Aedes aegypti. The result showed highest ovicidal activities (LC50 value of 4.84 ppm at 72 h) of citral followed by larvicidal (LC50 value of 87.02 ppm at 24 h) and adulticidal (LC50 value of 103.88 ppm at 24 h) activities. CONCLUSION: From this study, it can be concluded that the essential oil extracted from the leaf and peel of Citrus aurantifolia and one of its major constituent compound citral can be included in the mosquito control programme of Aedes aegypti.

The Political, Research, Programmatic, and Social Responses to Adolescent Sexual and Reproductive Health and Rights in the 25 Years Since the International Conference on Population and Development.

Among the ground-breaking achievements of the International Conference on Population and Development (ICPD) was its call to place adolescent sexual and reproductive health (ASRH) on global health and development agendas. This article reviews progress made in low- and middle-income countries in the 25 years since the ICPD in six areas central to ASRH-adolescent pregnancy, HIV, child marriage, violence against women and girls, female genital mutilation, and menstrual hygiene and health. It also examines the ICPD's contribution to the progress made. The article presents epidemiologic levels and trends; political, research, programmatic and social responses; and factors that helped or hindered progress. To do so, it draws on research evidence and programmatic experience and the expertise and experiences of a wide number of individuals, including youth leaders, in numerous countries and organizations. Overall, looking across the six health topics over a 25-year trajectory, there has been great progress at the global and regional levels in putting adolescent health, and especially adolescent sexual and reproductive health and rights, higher on the agenda, raising investment in this area, building the epidemiologic and evidence-base, and setting norms to guide investment and action. At the national level, too, there has been progress in formulating laws and policies, developing strategies and programs and executing them, and engaging communities and societies in moving the agenda forward. Still, progress has been uneven across issues and geography. Furthermore, it has raced ahead sometimes and has stalled at others. The ICPD's Plan of Action contributed to the progress made in ASRH not just because of its bold call in 1994 but also because it provided a springboard for advocacy, investment, action, and research that remains important to this day.

Combinations of Plant Essential Oil Based Terpene Compounds as Larvicidal and Adulticidal Agent against Aedes aegypti (Diptera: Culicidae).

Insecticidal plant-based compound(s)in combinations may show synergistic or antagonistic interactions against insect pest. Considering the rapid spread of the Aedes borne diseases and increasing resistance among Aedes population against conventional insecticides, twenty-eight combinations of plant essential oil-based terpene compounds were prepared and tested against larval and adult stages ofAedes aegypti. Initially five plant essential oils (EOs) were assessed for their larvicidal and adulticidal efficacy and two of their major compounds from each EO were identified from GC-MS results. Identified major compounds namely Diallyldisulfide, Diallyltrisulfide, Carvone, Limonene, Eugenol, Methyl Eugenol, Eucalyptol, Eudesmol and α-pinene were purchased and tested individually against A. aegypti. Binary combinations of these compounds were then prepared using sub-lethal doses, tested and their synergistic and antagonistic effects were determined. The best larvicidal compositions were obtained while Limonene was mixed with Diallyldisulfide and the best adulticidal composition was obtained while Carvone was mixed with Limonene. Commercially used synthetic larvicide "Temephos" and adulticide "Malathion" were tested individually and in binary combinations with the terpene compounds. The results revealed that the combination of Temephos and Diallyldisulfide and combination of Malathion and Eudesmol were the most effective combination. These effective combinations bear potential prospect to be used against Aedes aegypti.