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Background: Treatment of diabetes mellitus (DM) using plant based drugs is advancing and getting much attention in recent years. Cassia auriculata L. is widely used in Indian folk medicine for the treatment of DM. Site-specific targeted plant drug delivery by metallic nanoparticles carriers is a new emerging procedure under research due to its enhanced bioavailability and reduced toxicity. This study was therefore aimed at biosynthesizing a new silver nanoparticle of Cassia auriculata L. flower extract and study its anti-diabetic efficiency in rats. Methods: The silver nanoparticles were biosynthesized using AgNO3 solution and characterized by spectroscopy, SEM and EDAX analyses. The acute toxicity of this nano-preparation (up to 2000 mg/kg b. wt) was analyzed in rats and the anti-diabetic efficiency (for 50 mg/kg.b.wt and 200 mg/kg.b.wt) was studied in Streptozotocin induced diabetic rats. The diabetic parameters such as; blood glucose, serum protein, liver glycogen, serum lipid profile, serum levels of creatinine, urea, ALT, AST and ALP were analyzed. The histology of the liver, kidney and pancreas was evaluated after staining with Haematoxylin and Eosin. Results: The increased blood glucose in Streptozotocin induced diabetic rats was found to be significantly reduced (p Conclusion: The results of this study, proving an efficient anti-diabetic activity of the biosynthesized silver nanoparticles of Cassia auriculata L. flower extract, implies that this nano-preparation may be exploited as an alternative biopharmaceutical agent for treating DM.

The molecular targeted drug ATRA demands a suitable carrier that delivers to the cancer site due to its poor bioavailability and drug resistance. ATRA, being a lipid with carboxylic acid, has been nano‐formulated as a cationic lipo‐ATRA with DOTAP:cholesterol:ATRA (5:4:1) and its pH‐responsive release, intracellular drug accumulation, and anticancer effect on human lung cancer (A549) cell line analysed. The analysis of the physicochemical characteristics of the developed lipo‐ATRA (0.8 µmol) revealed that the size of 231 ± 2.35 d.nm had a zeta potential of 6.4 ± 1.19 and an encapsulation efficiency of 93.7 ± 3.6%. The ATRA release from lipo‐ATRA in vitro was significantly (p ≤ 0.05) higher at acidic pH 6 compared to pH 7.5. The intracellular uptake of ATRA into lipo‐ATRA‐treated A549 cells was seven‐fold higher (0.007 ± 0.001 mg/ml) while only three‐fold uptake was observed in free ATRA treatment (0.003 ± 0.002 mg/ml). The lipo‐ATRA treatment caused a highly significant (p ≤ 0.001) decrease in percent cell viability at 48 h when compared with the free ATRA treatment. Overall, the results proved that the developed lipo‐ATRA has suitable physicochemical properties with enhanced ATRA release at acidic pH, while maintaining stability at physiologic pH and temperature. This resulted in an increased ATRA uptake by lung cancer cells with enhanced treatment efficiency. Hence, it is concluded that DOTAP lipo‐ATRA is a suitable carrier for ATRA delivery to solid cancer cells.

The changes that occur during metastasis lodging is under intense research now to develop preventive new drugs to fight against the deadly metastasis. The molecular drug, all trans Retinoic Acid (ATRA) has regulatory effects on signal mediated metabolism. In this study, we have analyzed the metastasis facilitating metabolic changes in mice lung when a highly metastatic melanoma cell line (B16F10) having potency to lodge in lung was implanted via tail vein injection into C57BL/6 mice (1×106 cells/ml in PBS). One group of implanted mice were treated with 0.60 mg of ATRA per Kg body weight daily for 21 days. The alteration of protein, enzymatic and non-enzymatic antioxidants (SOD, Catalase, GPX, GSH) levels and the lipid profile with cholesterol level were evaluated in the lung tissues. The ATRA treatment caused 62.16% inhibition on metastatic nodule formation. Compared to normal mice, the cancer control mice showed an increased (p≤ 0.01**) total protein, LPO and NO and a decreased antioxidant. In ATRA treated group, all these levels were reverted to near normal levels with a high significance (p≤ 0.01**) difference from untreated cancer mice. The lipid profile and cholesterol level also were altered in cancer and were normalized in ATRA treated group with high significance (p≤ 0.01**). All these results implies that the metabolic changes induced in the lung tissue during metastatic lodging of melanoma cells were prevented and regularized by the ATRA treatment in vivo which give a scope of anti-metastatic therapy using ATRA.

Lung cancer is the leading cancer according to the World Health Organization (WHO), resulting in highest death rate worldwide due to the high level of metastasis. Hence, the drugs that protect from metastasis either as an adjuvant or a primary therapeutic agent may help to reduce the death rate. In this study, All Trans Retinoic Acid (ATRA) was tested for its action against metastatic lodging of B16F10 melanoma cells in the lung and liver of the C57BL/6 mouse model. Serum, lung and liver were evaluated biochemically for the cancer associated changes. Metastatic cancer development was confirmed by tumor nodule formation and histopathological analysis. RAR-β protein expression was analyzed by immunohistochemistry and histopathology. ATRA treated mice showed a percentage of inhibition on metastatic tumor growth in lung and liver and a corresponding protection against pathological changes in these organs. Cholesterol and γ-Glutamyl Transferase (GGT) levels found in cancer induced mice were reduced in the ATRA treated group. As compared to the normal group, lung tissue from cell line induced cancer control group had less RAR-β protein expression while the ATRA treated group showed enhanced RAR-β protein expression. This indicates that the anti-metastasis effects of ATRA might have shown the induction of RAR-β expression and subsequent molecular signaling pathways to regulate the homeostasis of biochemical changes. This study demonstrated the capability of ATRA to prevent the establishment of metastasis by the melanoma cell line into the lung and liver of experimental mice.

Electrostatic protein/DNA interactions arise from the neutralization of the DNA phosphodiester backbone as well as coupled exchanges by charged protein residues as salt bridges or with mobile ions. Much focus has been and continues to be paid to interfacial ion pairs with DNA. The role of extra-interfacial ionic interactions, particularly as dynamic drivers of DNA sequence selectivity, remain poorly known. The ETS family of transcription factors represents an attractive model for addressing this knowledge gap given their diverse ionic composition in primary structures that fold to a tightly conserved DNA-binding motif. To probe the importance of extra-interfacial salt bridges in DNA recognition, we compared the salt-dependent binding by Elk1 with ETV6, two ETS homologs differing markedly in ionic composition. While both proteins exhibit salt-dependent binding with cognate DNA that corresponds to interfacial phosphate contacts, their nonspecific binding diverges from cognate binding as well as each other. Molecular dynamics simulations in explicit solvent, which generated ionic interactions in agreement with the experimental binding data, revealed distinct salt-bridge dynamics in the nonspecific complexes formed by the two proteins. Impaired DNA contact by ETV6 resulted in fewer backbone contacts in the nonspecific complex, while Elk1 exhibited a redistribution of extra-interfacial salt bridges via residues that are non-conserved between the two ETS relatives. Thus, primary structure variation in ionic residues can encode highly differentiated specificity mechanisms in a highly conserved DNA-binding motif.

This report describes a breakthrough in a project to design minor groove binders to recognize any sequence of DNA. A key goal is to invent synthetic chemistry for compound preparation to recognize an adjacent GG sequence that has been difficult to target. After trying several unsuccessful compound designs, an N-alkyl-benzodiimidazole structure was selected to provide two H-bond acceptors for the adjacent GG-NH groups. Flanking thiophenes provide a preorganized structure with strong affinity, DB2831, and the structure is terminated by phenyl-amidines. The binding experimental results for DB2831 with a target AAAGGTTT sequence were successful and include a high ΔTm, biosensor SPR with a KD of 4 nM, a similar KD from fluorescence titrations and supporting competition mass spectrometry. MD analysis of DB2831 bound to an AAAGGTTT site reveals that the two unprotonated N of the benzodiimidazole group form strong H-bonds (based on distance) with the two central G-NH while the central –CH of the benzodiimidazole is close to the –C Created by potrace 1.16, written by Peter Selinger 2001-2019 O of a C base. These three interactions account for the strong preference of DB2831 for a -GG- sequence. Surprisingly, a complex with one dynamic, interfacial water is favored with 75% occupancy., This report describes a breakthrough in a project to design minor groove binders to recognize any sequence of DNA.

This study focused on evaluating the potency of Methyl Palmitate in reducing in vivo toxicity with enhancement of anti-cancer effects of Sorafenib. In vitro anti-cancer effects on human Hep-G2 cell line were analysed by MTT, Trypan blue, clonogenic, wound scratch migration and TUNEL assays. An in vivo study for anti-angiogenesis effect, toxicity and teratogenicity was analysed in Zebrafish embryos. The combination of Sorafenib (4.5 µmol/L) with Methyl Palmitate (3 mmol/L) significantly enhanced anti-cancer effects on Hep-G2 cell line by increasing cytotoxicity (P ≤ .05 in MTT assay; P ≤ .01 in Trypan blue assay), apoptosis (P ≤ .05) and decreasing the metastatic migration (P ≤ .01) than Sorafenib alone treatment. A prominent inhibition of angiogenesis in vivo was observed for combination treatment. At 5 dpf, only

Introduction Ceftazidime–avibactam combines the established anti-pseudomonal cephalosporin, ceftazidime, with the novel non-β-lactam β-lactamase inhibitor, avibactam. Objectives The aim of this study was to evaluate the safety of ceftazidime–avibactam in adults using pooled data from two phase II (NCT00690378, NCT00752219) and five phase III (NCT01499290, NCT01726023, NCT01644643, NCT01808093 and NCT01595438/NCT01599806) clinical studies. Methods Safety data from seven multicentre, randomised, active-comparator studies were pooled by study group at the patient level for descriptive analyses, comprising patients with complicated urinary tract infection (cUTI), including pyelonephritis, complicated intra-abdominal infection (cIAI), or nosocomial pneumonia (NP), including ventilator-associated pneumonia (VAP), treated with ceftazidime–avibactam ± metronidazole or comparator. Results In total, 4050 patients (ceftazidime–avibactam ± metronidazole, n = 2024; comparator, n = 2026) were included in the pooled analysis. Adverse events (AEs) up to the last study visit occurred in 996 (49.2%) and 965 (47.6%) patients treated with ceftazidime–avibactam ± metronidazole and comparator, respectively. The most common AEs across treatment groups were diarrhoea, nausea, headache, vomiting and pyrexia. There were few discontinuations due to AEs (2.5% and 1.7% for ceftazidime–avibactam ± metronidazole and comparators, respectively). Overall rates of serious AEs were 8.7% for ceftazidime–avibactam ± metronidazole and 7.2% for comparators; respective rates of AEs with an outcome of death were 2.0% and 1.8%. AEs considered causally related to the study drug or procedures occurred in 10.7% and 9.6% of patients treated with ceftazidime–avibactam ± metronidazole and comparators; the most common drug-related AEs in both groups were diarrhoea, headache, nausea and increased alanine aminotransferase. No impact to the safety profile of ceftazidime–avibactam ± metronidazole was found with regard to intrinsic factors, such as age or renal function at baseline, or extrinsic factors, such as geographical origin. Potentially clinically significant changes in laboratory parameters were infrequent with no trends or safety concerns identified. Conclusion The observed safety profile of ceftazidime–avibactam across infection types is consistent with the established safety profile of ceftazidime monotherapy and no new safety findings were identified. This analysis supports the use of ceftazidime–avibactam as a treatment option in adults with cUTI, cIAI and NP, including VAP. Electronic supplementary material The online version of this article (10.1007/s40264-020-00934-3) contains supplementary material, which is available to authorized users.

The majority of current drugs against diseases, such as cancer, can bind to one or more sites in a protein and inhibit its activity. There are, however, well known limits on the number of druggable proteins and complementing current drugs with compounds that could selectively target DNA or RNA would greatly enhance therapeutic progress and options. We are focusing on the design of sequence-specific DNA minor groove binders that, for example, target the promoter sites of transcription factors involved in a disease. We have started with AT specific minor groove binders that are known to enter human cells and have entered clinical trials. To broaden the sequence-specific recognition of these compounds, we have identified several modules that have H-bond acceptors that strongly and specifically recognize G•C base pairs. A lead module is a thiophene-N-alkyl-benzimidazole σ-hole based system with terminal phenyl-amidines where the optimum compounds have excellent affinity and selectivity for a G•C base pair in the minor groove. Efforts are now focused on optimizing this module. We have previously optimized the alkyl group. In the work described here we are evaluating modifications to the compound aromatic system with the goal of improving GC selectivity and affinity as with the N-alkyl modifications. The lead compounds from these studies retain the thiophene-N-alkyl-BI module but have halogen substituents adjacent to an amidine group on the terminal phenyl-amidine. Other improved compounds in this set have modified amidines and conversion of the amidine to an imidazoline, for example, resulted in a strong binding compound with good specificity.

A novel series of small size diamidines with thiophene and modified N-alkylbenzimidazole σ-hole module represent specific binding to single G·C base pair (bp) DNA sequence. The variation of N-alkyl or aromatic rings were sensitive to microstructure of DNA minor groove. Thirteen new compounds were synthesized to test their binding affinity and selectivity. The dicyanobenzimidazoles needed to synthesize the target diamidines were made via condensation/cyclization reactions of different aldehydes with different 3-amino-4-(alkyl- or phenyl-amino) benzonitriles. The final diamidines were synthesized using lithium bis-trimethylsilylamide (LiN[Si(CH(3))(3)](2)) or Pinner methods. The newly synthesized compounds showed strong binding and selectivity to AAAGTTT compared to similar sequences AAATTT and AAAGCTTT investigated by several biophysical methods including biosensor-SPR, fluorescence spectroscopy, DNA thermal melting, ESI-MS spectrometry, Circular dichroism, and Molecular Dynamics. The binding affinity results determined by fluorescence spectroscopy are in accordance with those obtained by biosensor-SPR. These small size single G·C bp highly specific binders extend the compound database for future biological applications.

Hairpin polyamides are synthetic small molecules that bind DNA minor groove sequence-selectively and, in many sequences, induce widening of the minor groove and compression of the major groove. The structural distortion of DNA caused by polyamides has enhanced our understanding of the regulation of DNA-binding proteins via polyamides. Polyamides have DNA binding affinities that are comparable to those proteins, therefore, can potentially be used as therapeutic agents to treat diseases caused by aberrant gene expression. In fact, many diseases are characterized by over- or under-expressed genes. PU.1 is a transcription factor that regulates many immune system genes. Aberrant expression of PU.1 has been associated with the development of acute myeloid leukemia (AML). We have, therefore, designed and synthesized ten hairpin polyamides to investigate their capacity in controlling the PU.1-DNA interaction. Our results showed that nine of the polyamides disrupt PU.1-DNA binding and the inhibition capacity strongly correlates with binding affinity. One molecule, FH1024, was observed forming a FH1024-PU.1-DNA ternary complex instead of inhibiting PU.1-DNA binding. This is the first report of a small molecule that is potentially a weak agonist that recruits PU.1 to DNA. This finding sheds light on the design of polyamides that exhibit novel regulatory mechanisms on protein-DNA binding.

ion of a pyridine ligand from Weiss’ reagent ([PhI(Pyr)2]2+) using BF3-Et2O was found to activate Weiss’ reagent towards electrophilic aromatic substitution reactions. The activated species can be isolated when 4-DMAP is used as the pyridine ligand and was determined to be a [PhI(4-DMAP)]2+ in solution, and is associated with a triflate counterion in the solid state. The isolated cation was reactive in electrophilic aromatic substitution reactions towards mesitylene, xylene and toluene that Weiss’ reagent itself does not react with. If pyridine is used as the ligand the intermediate that results from abstraction cannot be isolated but is even more active, being observed to react with benzene.

Aim Inflammation provides favourable microenvironment for cancer development. An enhanced COX-2 gene expression is a key inflammatory mediator of cancers and the drug that inhibits it, helps to manage cancer effectively and increases survival rate. The objective is to analyse the inflammatory changes and COX-2 gene expression in benzo (a) pyrene induced mice and to evaluate the regulatory effect of all trans retinoic acid. Materials and methods The body and organ weights were recorded in B(a)P induced mice. The haematological parameters and serum inflammatory markers of carcinogenesis were tested. The H & E stained liver and lung tissues were examined for histopathologic changes. The COX-2 gene expression was analysed by RT-PCR and qPCR in lung and liver. Key findings The decreased body weight, increased organ weights and the damages in liver and lung were observed in B(a)P induced mice and were prevented significantly upon ATRA treatment. The lowered Hb, RBC and lymphocytes and an enhanced WBC, monocytes and neutrophils observed in B(a)P group were significantly reversed in treated group. A drastic increase in cancer associated inflammatory markers observed in B(a)P induced mice were significantly (P ≤ 0.001) reduced in treated mice. The RT-PCR product density of COX-2 gene was very high in B(a)P group (lung-0.43 ± 0.06; liver-0.39 ± 0.04) significantly lower in treated group (lung-0.12 ± 0.03; liver-0.08 ± 0.03) with a significant difference in RQ values (B(a)P lung-18.46 ± 0.04, liver-12.46 ± 0.08; treated lung-5.93 ± 0.07, liver-2.92 ± 0.10). Significance The ATRA has decreased the inflammatory condition with downregulation of COX-2 gene expression and thereby prevented carcinogenesis during early stage of B(a)P induced cancer development.

Titanium alloys, in particular, medical-grade Ti-6Al-4 V, are heavily used in orthopaedic applications due to their high moduli, strength, and biocompatibility. Implant infection can result in biofilm formation and failure of prosthesis. The formation of a biofilm on implants protects bacteria from antibiotics and the immune response, resulting in the propagation of the infection and ultimately resulting in device failure. Recently, slippery liquid-infused surfaces (LIS) have been investigated for their stable liquid interface, which provides excellent repellent properties to suppress biofilm formation. One of the current limitations of LIS coatings lies in the indistinctive repellency of bone cells in orthopaedic applications, resulting in poor tissue integration and bone ingrowth with the implant. Here, we report a chitosan impregnated LIS coating that facilitates cell adhesion while preventing biofilm formation. The fabricated coating displayed high contact angles (108.2 ± 5.2°) and low sliding angles (3.56 ± 4.3°). Elemental analysis obtained using X-ray photoelectron spectroscopy (XPS) confirmed the availability of fluorine and nitrogen, indicating the presence of fluorosilane and chitosan in the final coating. Furthermore, our results suggest that chitosan-conjugated LIS increased cell adhesion of osteoblast-like SaOS-2 cells and significantly promoted proliferation (a fourfold increase at 7-day incubation) compared to conventional titanium liquid-infused surfaces. Furthermore, the chitosan conjugated LIS significantly reduced biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) by up to 50% and 75% when compared to untreated titanium and chitosan-coated titanium, respectively. The engineered coating can be easily modified with other biopolymers or capture molecules to be applied to other biomaterials where tissue integration and biofilm prevention are needed.

A study on the potential activating role of pyridine in the electrophilic chlorination of anisole by PhICl2 has led to the discovery that soluble sources of chloride ions activate PhICl2 in the reaction at catalytic loadings, greatly increasing the rate of chlorination. It is further shown that presence of chloride increases the rate of decomposition of PhICl2 into PhI and Cl2. The specific mechanism by which chloride induces electrophilic chlorination and decomposition of PhICl2 remains an open question.

Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well-studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes.

Emissions of various fuel components (cyclohexane, ethanol, and pentane) and reaction intermediate species (acetylene, ethylene, formaldehyde, and methane) from a multicylinder, port-fuel-injected,...

Most transcription factors were for a long time considered as undruggable targets because of the absence of binding pockets for direct targeting. HOXA9, implicated in acute myeloid leukemia, is one of them. To date, only indirect targeting of HOXA9 expression or multitarget HOX/PBX protein/protein interaction inhibitors has been developed. As an attractive alternative by inhibiting the DNA binding, we selected a series of heterocyclic diamidines as efficient competitors for the HOXA9/DNA interaction through binding as minor groove DNA ligands on the HOXA9 cognate sequence. Selected DB818 and DB1055 compounds altered HOXA9-mediated transcription in luciferase assays, cell survival, and cell cycle, but increased cell death and granulocyte/monocyte differentiation, two main HOXA9 functions also highlighted using transcriptomic analysis of DB818-treated murine Hoxa9-transformed hematopoietic cells. Altogether, these data demonstrate for the first time the propensity of sequence-selective DNA ligands to inhibit HOXA9/DNA binding both in vitro and in a murine Hoxa9-dependent leukemic cell model.

In this research the effect of two fundamental stresses, i.e. the tableting load and roller compaction pressure, on tablet disintegration were investigated. Excipient materials were roller compacted using a range of pressures, and tablets were produced by compressing the consequent granules at varying tableting loads. For this study mannitol, a brittle and soluble material, and microcrystalline cellulose, a deformable and insoluble material, were investigated at varying binary ratios. The tablets were characterised by analysis of the compactibility and a flow cell imaging method was utilised to investigate tablet disintegration in real-time. It was found that the effect of roller compaction (RC) pressure on tablet tensile strength and porosity was dependent on the tableting load used. At low tableting load, the tablet porosity varied depending on the RC pressure used, whereas the tensile strength was largely unaffected. The use of a high tableting load leads to the opposite effect being observed. The tensile strength was highly dependent on the RC pressure used. In terms of the tablet disintegration the RC pressure did not influence the disintegration behaviour when low tableting loads were used as the process was rapid. When higher tableting loads were used, tablets containing granules roller compacted using lower pressure expanded in size larger than when a low RC pressure was used. A low RC pressure also lead to a faster particle release rate was observed.

Dicationic diamidines have been well established as potent antiparasitic agents with proven activity against tropical diseases like trypanosomiasis and malaria. This work presents the synthesis of new mono and diflexible triaryl amidines (6a-c, 13a,b and 17), their aza analogues (23 and 27) and respective methoxyamidine prodrugs (5, 7, 12a,b, 22 and 26). All diamidines were assessed in vitro against Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.) where they displayed potent to moderate activities at the nanomolar level with IC50s = 11–378 nM for T. b. r. and 4–323 nM against P. f.. In vivo efficacy testing against T. b. r. STIB900 has shown the monoflexible diamidine 6c as the most potent derivative in this study eliciting 4/4 cures of infected mice for a treatment period of >60 days upon a 4 × 5 mg/kg dose i. p. treatment. Moreover, thermal melting analysis measurement ΔTm for this series of diamidines/poly (dA-dT) complexes fell between 0.5 and 19 °C with 6c showing the highest binding to the DNA minor groove. Finally, a 50 ns molecular dynamics study of an AT-rich DNA dodecamer with compound 6c revealed a strong binding complex supported by vdW and electrostatic interactions.

International audience; Cone snails are venomous marine predators that rely on fast-acting venom to subdue their prey and defend against aggressors. The conotoxins produced in the venom gland are small disulfide-rich peptides with high affinity and selectivity for their pharmacological targets. A dominant group comprises α-conotoxins, targeting nicotinic acetylcholine receptors. Here, we report on the synthesis, structure determination and biological activity of a novel α-conotoxin, CIC, found in the predatory venom of the piscivorous species Conus catus and its truncated mutant Δ-CIC. CIC is a 4/7 α-conotoxin with an unusual extended N-terminal tail. High-resolution NMR spectroscopy shows a major influence of the N-terminal tail on the apparent rigidity of the three-dimensional structure of CIC compared to the more flexible Δ-CIC. Surprisingly, this effect on the structure does not alter the biological activity, since both peptides selectively inhibit α3β2 and α6/α3β2β3 nAChRs with almost identical sub- to low micromolar inhibition constants. Our results suggest that the N-terminal part of α-conotoxins can accommodate chemical modifications without affecting their pharmacology.

Mapping-by-sequencing strategies combine next-generation sequencing (NGS) with classical linkage analysis, allowing rapid identification of the causal mutations of the phenotypes exhibited by mutants isolated in a genetic screen. Computer programs that analyze NGS data obtained from a mapping population of individuals derived from a mutant of interest in order to identify a causal mutation are available; however, the installation and usage of such programs requires bioinformatic skills, modifying or combining pieces of existing software, or purchasing licenses. To ease this process, we developed Easymap, an open-source program that simplifies the data analysis workflows from raw NGS reads to candidate mutations. Easymap can perform bulked segregant mapping of point mutations induced by ethyl methanesulfonate (EMS) with DNA-seq or RNA-seq datasets, as well as tagged-sequence mapping for large insertions, such as transposons or T-DNAs. The mapping analyses implemented in Easymap have been validated with experimental and simulated datasets from different plant and animal model species. Easymap was designed to be accessible to all users regardless of their bioinformatics skills by implementing a user-friendly graphical interface, a simple universal installation script, and detailed mapping reports, including informative images and complementary data for assessment of the mapping results.One sentence summaryEasymap is a versatile user-friendly software tool that facilitates mapping-by-sequencing of large insertions and point mutations in plant and animal genomes

Two horses that consumed well water with high fluoride content exhibited clinical signs of chronic dental and skeletal fluoride toxicosis and were later euthanized and autopsied. Both horses had degenerative disease of multiple joints and multiple dental defects. Elevated fluoride concentrations were found in bone and tooth samples of both horses, well water, and feed. Microscopically, abnormalities were noted in bone and tooth samples, and consisted mostly of foci of cement necrosis and hypercementosis. Horses exhibiting bilateral, highly symmetrical dental and/or skeletal lesions, with chronic lameness, should be evaluated for the possible presence of fluoride toxicosis.

[Image: see text] Targeting of genomic quadruplexes is an approach to treating complex human cancers. We describe a series of tetra-substituted naphthalene diimide (ND) derivatives with a phenyl substituent directly attached to the ND core. The lead compound (SOP1812) has 10 times superior cellular and in vivo activity compared with previous ND compounds and nanomolar binding to human quadruplexes. The pharmacological properties of SOP1812 indicate good bioavailability, which is consistent with the in vivo activity in xenograft and genetic models for pancreatic cancer. Transcriptome analysis shows that it down-regulates several cancer gene pathways, including Wnt/β-catenin signaling.

DB2277, a heterocyclic diamidine, is a successful design for mixed base pair (bp) DNA sequence recognition. The compound has a central aza-benzimidazole group that forms two H-bonds with a GC bp that has flanking AT bps. The nuclear magnetic resonance structure of the DB2277-DNA complex with an AAGATA recognition site sequence was determined, and here we report extended molecular dynamics (MD) simulations of the structure. DB2277 has two terminal phenyl-amidine groups, one of which is directly linked to the DB2277 heterocyclic core and the other through a flexible -OCH2- group. The flexibly linked phenyl is too far from the minor groove floor to make direct H-bonds but is linked to an AT bp through water-mediated H-bonds. The flexibly linked phenyl-amidine with water-mediated H-bonds to the bases at the floor of the minor groove suggested that it might rotate in time spans accessible in MD. To test this idea, we conducted multimicrosecond MD simulations to determine if these phenyl rotations could be observed for a bound compound. In a 3 μs simulation, highly dynamic torsional motions were observed for the -OCH2-linked phenyl but not for the other phenyl. The dynamics periodically reached a level to allow 180° rotation of the phenyl while it was still bound in the minor groove. This is the first observation of rotation of a phenyl bound to DNA, and the results provide mechanistic details about how a rotation can occur as well as how mixed bp recognition can occur for monomer compounds bound to the minor groove.

The Diavik Waste Rock Project consists of laboratory and field experiments developed for the investigation and scale-up of the geochemical evolution of sulfidic mine wastes. As part of this project, humidity cell experiments were conducted to assess the long-term geochemical evolution of a low-sulfide waste rock. Reactive transport modelling was used to assess the significant geochemical processes controlling oxidation of sulfide minerals and their dependence on temperature and sulfide mineral content. The geochemical evolution of effluent from waste rock with a sulfide content of 0.16 wt.% and 0.02 wt.% in humidity cells was simulated with the reactive transport model MIN3P, based on a conceptual model that included constant water flow, sulfide mineral content, sulfide oxidation controlled by the availability of oxidants, and subsequent neutralization reactions with carbonate and aluminosilicate minerals. Concentrations of Ni, Co, Cu, Zn, and SO4 in the humidity cell effluent were simulated using the shrinking core model, which represented the control of oxidant diffusion to the unreacted particle surface in the sulfide oxidation process. The influence of temperature was accounted for using the Arrhenius relation and appropriate activation energy values. Comparison of the experiment results, consisting of waste rock differentiated by sulfide mineral content and temperature, indicated surface area and temperature play important roles in rates of sulfide oxidation and release of sulfate and metals. After the model was calibrated to fit the effluent data from the higher sulfide content cells, subsequent simulations were conducted by adjusting only measured parameters, including sulfide mineral content and surface area.

To extend the value of biosensor-SPR in the characterization of DNA recognition by nucleoproteins, we report a comparative analysis of DNA-facilitated target search by two ETS-family transcription factors: Elk1 and ETV6. ETS domains represent an attractive system for developing biosensor-based techniques due to a broad range of physicochemical properties encoded within a highly conserved DNA-binding motif. Building on a biosensor approach in which the protein is quantitatively sequestered and presented to immobilized cognate DNA as nonspecific complexes, we assessed the impact of intrinsic cognate and nonspecific affinities on long-range (intersegmental) target search. The equilibrium constants of DNA-facilitated binding were sensitive to the intrinsic binding properties of the proteins such that their relative specificity for cognate DNA were reinforced when binding occurred by transfer vs. without nonspecific DNA. Direct measurement of association and dissociation kinetics revealed ionic features of the activated complex that evidenced DNA-facilitated dissociation, even though Elk1 and ETV6 harbor only a single DNA-binding surface. At salt concentrations that masked the effects of nonspecific pre-binding at equilibrium, the dissociation kinetics of cognate binding were nevertheless distinct from conditions under which nonspecific DNA was absent. These results further strengthen the significance of long-range DNA-facilitated translocation in the physiologic environment.

Cellulase activity on insoluble cellulose substrates declines as the substrate is modified. The role of structural changes that result in substrate recalcitrance, such as changes to cellulose crystallinity, requires further investigation. Crystallinity of cellulose samples with varying extents of digestion can only be compared meaningfully using a high throughput - Fourier transform infrared spectroscopy (HTS-FTIR) technique when the many variables involved are carefully controlled. Hence, changes to the HTS-FTIR sample preparation methods previously described in literature were necessary in order to obtain clean raw spectra and reliable measures of cellulose crystallinity. The sample preparation methods of residual cellulose after digestion by individual cellulases and a complex cellulase mixture from T. fusca were improved to remove extraneous overlapping signals, provide accurate extent of digestion, and correct errors caused by varying concentrations. These improved preparation methods enabled measurement of crystallinity index values of residual cellulose which did not show a correlation between cellulose crystallinity and the decline in cellulase activity.

Characterisation of anodic aluminium oxide coatings and measurement of their thickness using microscopic techniques is valuable for analysing the effectiveness of the prior anodising process. Three different methods for preparing samples to view the coating cross-section (mechanical fracturing, cryogenic fracturing and metallography) were trialled and assessed for speed of implementation, simplicity and achievable measurement accuracy. Cryogenic fracturing was found to be destructive to samples. Mechanical fracturing yielded relatively accurate coating thickness measurements and coating structural information. Metallography provided the most accurate coating thickness measurement at the expense of coating structural information.

YM155 (sepantronium bromide) has been evaluated in clinical trials as a survivin suppressant, but despite positive signals from early work, later studies were negative. Clarification of the mechanism of action of YM155 is important for its further development. YM155 affects cells in a cell cycle-specific manner. When cells are in G1, YM155 prevented their progression through the S phase, leaving the cells at G1/S when exposed to YM155. Passage through mitosis from G2 is also defective following YM155 exposure. In this study, YM155 did not behave like a typical DNA intercalator in viscosity, circular dichroism, and absorption spectroscopy studies. In addition, molecular modeling experiments ruled out YM155 DNA interaction to produce DNA intercalation. We show that YM155 inhibited topoisomerase 2α decatenation and topoisomerase 1-mediated cleavage of DNA, suggesting that YM155 inhibits the enzyme function. Consistent with these findings, DNA double-strand break repair was also inhibited by YM155.

Macrophages orchestrate the initiation and resolution of inflammation by producing pro- and anti-inflammatory products. An imbalance in these mediators may originate from a deficient or excessive immune response. Therefore, macrophages are valid therapeutic targets to restore homeostasis under inflammatory conditions. We hypothesize that a specific mannosylated nanoparticle effectively induces gene expression in human macrophages under inflammatory conditions without undesirable immunogenic responses. THP-1 macrophages were challenged with lipopolysaccharide (LPS, 5 μg/mL). Polyethylenimine (PEI) nanoparticles grafted with a mannose receptor ligand (Man-PEI) were used as a gene delivery method. Nanoparticle toxicity, Man-PEI cellular uptake rate and gene induction efficiency (GFP, CD14 or CD68) were studied. Potential immunogenic responses were evaluated by measuring the production of tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-10. Man-PEI did not produce cytotoxicity, and it was effectively up-taken by THP-1 macrophages (69%). This approach produced a significant expression of GFP (mRNA and protein), CD14 and CD68 (mRNA), and transiently and mildly reduced IL-6 and IL-10 levels in LPS-challenged macrophages. Our results indicate that Man-PEI is suitable for inducing an efficient gene overexpression in human macrophages under inflammatory conditions with limited immunogenic responses. Our promising results set the foundation to test this technology to induce functional anti-inflammatory genes.

Minor-groove binding hairpin polyamides (PAs) bind specific DNA sequences. Synthetic modifications can improve PA-DNA binding affinity and include flexible modules, such as β-alanine (β) motifs to replace pyrroles (Py), and increasing compound charge using N-terminal cationic substituents. To better understand the variations in kinetics and affinities caused by these modifications on PA-DNA interactions, a comprehensive set of PAs with different numbers and positions of β and different types of N-cationic groups was systematically designed and synthesized to bind their cognate sequence, the λB motif. The λB motif is also a strong binding promoter site of the major groove targeting transcription factor PU.1. The PA binding affinities and kinetics were evaluated using a spectrum of powerful biophysical methods: thermal melting, biosensor surface plasmon resonance and circular dichroism. The results show that β inserts affect PA-DNA interactions in a number and position dependent manner. Specifically, a β replacement between two imidazole heterocycles (ImβIm) generally strengthens binding. In addition, N-terminal cationic groups can accelerate the association between PA and DNA, but the bulky size of TMG can cause steric hindrance and unfavourable repulsive electrostatic interactions in some PAs. The future design of stronger binding PA requires careful combination of βs and cationic substituents.

The design and synthesis of compounds that target mixed, AT/GC, DNA sequences is described. The design concept connects two N-methyl-benzimidazole-thiophene single GC recognition units with a flexible linker that lets the compound fit the shape and twist of the DNA minor groove while covering a full turn of the double helix.

Specific targeting of protein–nucleic acid interactions is an area of current interest, for example, in the regulation of gene-expression. Most transcription factor proteins bind in the DNA major groove; however, we are interested in an approach using small molecules to target the minor groove to control expression by an allosteric mechanism. In an effort to broaden sequence recognition of DNA-targeted-small-molecules to include both A·T and G·C base pairs, we recently discovered that the heterocyclic diamidine, DB2277, forms a strong monomer complex with a DNA sequence containing 5΄-AAAGTTT-3΄. Competition mass spectrometry and surface plasmon resonance identified new monomer complexes, as well as unexpected binding of two DB2277 with certain sequences. Inherent microstructural differences within the experimental DNAs were identified through computational analyses to understand the molecular basis for recognition. These findings emphasize the critical nature of the DNA minor groove microstructure for sequence-specific recognition and offer new avenues to design synthetic small molecules for effective regulation of gene-expression.

G-Quadruplex DNA has been recognized as a highly appealing target for the development of new selective chemotherapeutics, which could result in markedly reduced toxicity toward normal cells. In particular, the cyanine dyes that bind selectively to G-quadruplex structures without targeting duplex DNA have attracted attention due to their high amenability to structural modifications that allows fine-tuning of their biomolecular interactions. We have previously reported pentamethine and symmetric trimethine cyanines designed to effectively bind G-quadruplexes through end stacking interactions. Herein, we are reporting a second generation of drug candidates, the asymmetric trimethine cyanines. These have been synthesized and evaluated for their quadruplex binding properties. Incorporating a benz[c,d]indolenine heterocyclic unit increased overall quadruplex binding, and elongating the alkyl length increases the quadruplex-to-duplex binding specificity.

IntroductionEswatini achieved a 44% decrease in new HIV infections from 2014 to 2019 through substantial scale-up of testing and treatment. However, it still has one of the highest rates of HIV incidence in the world, with 14 infections per 1,000 adults 15-49 years estimated for 2017. The Government of Eswatini has called for an 85% reduction in new infections by 2023 over 2017 levels. To make further progress towards this target and to achieve maximum health gains, this study aims to model optimized investments of available HIV resources.MethodsThe Optima HIV model was applied to estimate the impact of efficiency strategies to accelerate prevention of HIV infections and HIV-related deaths. We estimated the number of infections and deaths that could be prevented by optimizing HIV investments. We optimize across HIV programs, then across service delivery modalities for voluntary medical male circumcision (VMMC), HIV testing, and antiretroviral refill, as well as switching to a lower cost antiretroviral regimen.FindingsUnder an optimized budget, prioritising HIV testing for the general population followed by key preventative interventions may result in approximately 1,000 more new infections (2% more) being averted by 2023. More infections could be averted with further optimization between service delivery modalities across the HIV cascade. Scaling-up index and self-testing could lead to 100,000 more people getting tested for HIV (25% more tests) with the same budget. By prioritizing Fast-Track, community-based, and facility-based antiretroviral refill options, an estimated 30,000 more people could receive treatment, 17% more than baseline or US$5.5 million could be saved, 4% of the total budget. Finally, switching non-pregnant HIV-positive adults to a Dolutegravir-based antiretroviral therapy regimen and concentrating delivery of VMMC to existing fixed facilities over mobile clinics, US$4.5 million (7% of total budget) and US$6.6 million (10% of total budget) could be saved, respectively.SignificanceWith a relatively short five-year timeframe, even under a substantially increased and optimized budget, Eswatini is unlikely to reach their ambitious national prevention target by 2023. However, by optimizing investment of the same budget towards highly cost-effective VMMC, testing, and treatment modalities, further reductions in HIV incidence and cost savings could be realized.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma driven by mutations in KIT or platelet-derived growth factor α (PDGFRα). Although first-line treatment, imatinib, has revolutionized GIST treatment, drug resistance due to acquisition of secondary KIT/PDGFRα mutations develops in a majority of patients. Second- and third-line treatments, sunitinib and regorafenib, lack activity against a plethora of mutations in KIT/PDGFRα in GIST, with median time to disease progression of 4 to 6 months and inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) causing high-grade hypertension. Patients with GIST have an unmet need for a well-tolerated drug that robustly inhibits a range of KIT/PDGFRα mutations. Here, we report the discovery and pharmacological characterization of AZD3229, a potent and selective small-molecule inhibitor of KIT and PDGFRα designed to inhibit a broad range of primary and imatinib-resistant secondary mutations seen in GIST. In engineered and GIST-derived cell lines, AZD3229 is 15 to 60 times more potent than imatinib in inhibiting KIT primary mutations and has low nanomolar activity against a wide spectrum of secondary mutations. AZD3229 causes durable inhibition of KIT signaling in patient-derived xenograft (PDX) models of GIST, leading to tumor regressions at doses that showed no changes in arterial blood pressure (BP) in rat telemetry studies. AZD3229 has a superior potency and selectivity profile to standard of care (SoC) agents-imatinib, sunitinib, and regorafenib, as well as investigational agents, avapritinib (BLU-285) and ripretinib (DCC-2618). AZD3229 has the potential to be a best-in-class inhibitor for clinically relevant KIT/PDGFRα mutations in GIST.

The leaves of traditionally used herbal plant Tridax procumbens L. contain lots of phytochemicals having potency to reduce inflammation. In this study, the ethanol extract of the leaves of Tridax procumbens L. was analysed for the phytochemicals by GC–MS. The anti-inflammatory activity was then studied with the extract of 10, 50, and 100 mg/kg b.wt in carrageenan-induced mice model by measuring the inflammatory oedema and by analysing the histopathology. The mRNA expression levels of TNF-α and COX2 genes were studied in the inflammatory site to explore the molecular action by reverse transcription PCR and qPCR analyses. A significant (P ≤ 0.01) reduction in mice paw inflammation and a recovered histology were observed in treated groups when compared to control group in 24 h. The RT-PCR results showed a significant (P ≤ 0.01) decrease in the expression levels of TNF-α and COX2 in terms of band density in treated mice compared to control group. The qPCR RQ values also were decreased in treated groups with respect to increasing doses (RQ values of 18.985 ± 0.230, 12.140 ± 1.121, 6.718 ± 0.807 for TNF-α and 15.583 ± 1.043, 7.725 ± 1.013, 5.075 ± 0.615 for COX2, respectively for the three doses) in comparison with the control group (TNF-α 27.107 ± 2.254, COX2 20.626 ± 1.477). Tridax procumbens L. can be, thus, used for the development of a safe, natural, anti-inflammatory drug as it showed a strong inhibitory action on inflammation by acting at molecular level.

The ends of nucleic acids oligomers alter the statistics of interior nonspecific ligand binding and act as binding sites with altered properties. While the former aspect of “end effects” has received much theoretical attention in the literature, the physical nature of end-binding, and hence its potential impact on a wide range of studies with oligomers, remains poorly known. Here, we report for the first time end-binding to DNA using a model helix-turn-helix motif, the DNA-binding domain of ETV6, as a function of DNA sequence length. Spectral analysis of ETV6 intrinsic tryptophan fluorescence by singular value decomposition showed that end-binding to nonspecific fragments was negligible at >0.2 kbp and accumulated to 8% of total binding to 23-bp oligomers. The affinity for end-binding was insensitive to salt but tracked the affinity of interior binding, suggesting translocation from interior sites rather than free solution as its mechanism. As the presence of a cognate site in the 23-bp oligomer suppressed end-binding, neglect of end-binding to the short cognate DNA does not introduce significant error. However, the same applies to nonspecific DNA only if longer fragments (>0.2 kbp) are used.

Chlorotoxin was originally isolated from the venom of the Israeli scorpion Leiurus quinquestriatus, and has potential as a tumor imaging agent based on its selective binding to tumor cells. Several targets have been suggested for chlorotoxin including voltage-gated chloride channels, and it has been shown to have anti-angiogenic activity and inhibit cell migration. The structure of chlorotoxin is stabilized by four disulfide bonds and contains β-sheet and helical structure. Interestingly, the reduced form has previously been shown to inhibit cell migration to the same extent as the wild type, but structural analysis indicates that the reduced form of the peptide does not maintain the native secondary structure and appears unstructured in solution. This lack of structure suggests that a short stretch of amino acids might be responsible for the bioactivity. To explore this hypothesis, we have synthesized fragments of chlorotoxin without disulfide bonds. As expected for such small peptides, NMR analysis indicated that the peptides were unstructured in solution. However, the peptide corresponding to the eight C-terminal residues inhibited cell migration, in contrast to the other fragments. Our results suggest that the C-terminal region plays a critical role in the bioactivity of chlorotoxin.

Regulation of gene-expression by specific targeting of protein–nucleic acid interactions has been a long-standing goal in medicinal chemistry. Transcription factors are considered “undruggable” because they lack binding sites well suited for binding small-molecules. In order to overcome this obstacle, we are interested in designing small molecules that bind to the corresponding promoter sequences and either prevent or modulate transcription factor association via an allosteric mechanism. To achieve this, we must design small molecules that are both sequence-specific and able to target G/C base pair sites. A thorough understanding of the relationship between binding affinity and the structural aspects of the small molecule–DNA complex would greatly aid in rational design of such compounds. Here we present a comprehensive analysis of sequence-specific DNA association of a synthetic minor groove binder using long timescale molecular dynamics. We show how binding selectivity arises from a combination of structural factors. Our results provide a framework for the rational design and optimization of synthetic small molecules in order to improve site-specific targeting of DNA for therapeutic uses in the design of selective DNA binders targeting transcription regulation.

Sequence-specific binding to DNA is crucial for targeting transcription factor-DNA complexes to modulate gene expression. The heterocyclic diamidine, DB2277, specifically recognizes a single G•C base pair in the minor groove of mixed base pair sequences of the type AAAGTTT. NMR spectroscopy reveals the presence of major and minor species of the bound compound. To understand the principles that determine the binding affinity and orientation in mixed sequences of DNA, over thirty DNA hairpin substrates were examined by NMR and thermal melting. The NMR exchange dynamics between major and minor species shows that the exchange is much faster than compound dissociation determined from biosensor–surface plasmon resonance. Extensive modifications of DNA sequences resulted in a unique DNA sequence with binding site AAGATA that binds DB2277 in a single orientation. A molecular docking result agrees with the model representing rapid flipping of DB2277 between major and minor species. Imino spectral analysis of a 15N-labeled central G clearly shows the crucial role of the exocyclic amino group of G in sequence-specific recognition. Our results suggest that this approach can be expanded to additional modules for recognition of more sequence-specific DNA complexes. This approach provides substantial information about the sequence-specific, highly efficient, dynamic nature of minor groove binding agents.

Organic soils are widespread in Ireland and vulnerable to degradation via drainage for agriculture. The soil-landuse combination of pasture on organic soils may play a disproportionate role in regional C dynamics but is yet to receive study. Fluvial C fluxes and labile organic fractions were determined for two such sites at nested field (c.4 ha) and subcatchment scales (>40 ha); one relatively dry and nutrient rich, the other wetter and nutrient poor. Field scale flux from the nutrient poor site over 2 years was 38.9 ± 6.6 g C m−2 yr−1 with DIC > DOC > POC at 57, 32 and 11 % respectively, and 72 % DIC was comprised of above equilibrium CO2. At the nutrient rich site, which overlies limestone geology, field scale export over an individual year was 90.4 g C m−2 with DIC > DOC > POC at 49, 42 and 9 %, but with 90 % DIC as bicarbonate. By comparison with the nutrient poor site, the magnitude and composition of inorganic C exports from the nutrient rich site implied considerable export of soil-respiratory C as bicarbonate, and lower evasion losses due to carbonate system buffering. Labile DOC determined using dark incubations indicated small fractions (5–10 %) available for remineralisation over typical downstream transit times of days to weeks. These fractions are probably conservative as photolysis in the environment can increase the proportion of labile compounds via photocleavage and directly remineralise organic matter. This study demonstrates that monitoring at soil–water interfaces can aid capture of total landscape fluvial fluxes by precluding the need to incorporate prior C evasion, although rapid runoff responses at field scales can necessitate high resolution flow proportional, and hydrograph sampling to constrain uncertainty of flux estimates.

Sustained oxidation of sulfide minerals in waste rock generates acid mine drainage for time frames of hundreds to thousands of years. Management to minimize long-term degradation of water supplies and ecosystem health requires a thorough understanding of geochemical processes occurring within the waste rock. A comprehensive field investigation was conducted on sulfide and carbonate-rich waste-rock dumps at the Faro Mine Complex, south-central Yukon Territory, Canada. This investigation included installation of three highly instrumented boreholes, in situ measurements of physical and geochemical parameters (e.g., water content, temperature, air permeability, and pore-gas O2 and CO2 concentration), and collection of pore-water, pore-gas, and solid-phase samples. Field and laboratory measurements indicate the waste rock is lithologically and mineralogically segregated, with sulfide-rich (>20 wt% S) waste rock dominating in the lower benches (20–50 mbgs) and carbonate-bearing waste rock dominating in the upper benches and other parts of the dumps. The observed segregation strongly influences spatial variability of geochemical parameters. Three distinct geochemical zones are observed within the waste-rock dumps, including a rapid O2-supply zone, with near-atmospheric concentrations of O2 (0–30 mbgs); a strong O2-depletion zone (30–50 mbgs), characterized by abundant sulfide minerals and significant O2 depletion to

Cyanine compounds have previously shown excellent in vitro and promising in vivo antileishmanial efficacy, but the potential toxicity of these agents is a concern. A series of 22 analogs of thiazole orange ((Z)-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium salt), a commercial cyanine dye with antileishmanial activity, were synthesized in an effort to increase the selectivity of such compounds while maintaining efficacy. Cyanines possessing substitutions on the quinolinium ring system displayed potency against Leishmania donovani axenic amastigotes that differed little from the parent compound (IC50 12–42 nM), while ring disjunction analogs were both less potent and less toxic. Changes in DNA melting temperature were modest when synthetic oligonucleotides were incubated with selected analogs (ΔTm ≤ 5 °C), with ring disjunction analogs showing the least effect on this parameter. Despite the high antileishmanial potency of the target compounds, their toxicity and relatively flat SAR suggests that further information regarding the target(s) of these molecules is needed to aid their development as antileishmanials.

AT specific heterocyclic cations that bind in the DNA duplex minor groove have had major successes as cell and nuclear stains and as therapeutic agents which can effectively enter human cells. Expanding the DNA sequence recognition capability of the minor groove compounds could also expand their therapeutic targets and have an impact in many areas, such as modulation of transcription factor biological activity. Success in the design of mixed sequence binding compounds has been achieved with N-methylbenzimidazole (N-MeBI) thiophenes which are preorganized to fit the shape of the DNA minor groove and H-bond to the –NH of G·C base pairs that projects into the minor groove. Initial compounds bind strongly to a single G·C base pair in an AT context with a specificity ratio of 50 (K(D) AT-GC/K(D) AT) or less and this is somewhat low for biological use. We felt that modifications of compound shape could be used to probe local DNA microstructure in target mixed base pair sequences of DNA and potentially improve the compound binding selectivity. Modifications were made by increasing the size of the benzimidazole N-substituent, for example, by using N-isobutyl instead of N-Me, and by changing the molecular twist by introducing substitutions at specific positions on the aromatic core of the compounds. In both cases, we have been able to achieve a dramatic increase in binding specificity, including no detectible binding to pure AT sequences, without a significant loss in affinity to mixed base pair target sequences.

Background The increased adverse cardiac events in women undergoing coronary artery bypass grafting are multifactorial and may include clinical, psychosocial, and biological factors. Potential contributing biological factors could include vascular hyperreactivity of the internal mammary artery ( IMA ) to endogenous vasoconstrictors in women, resulting in a predilection to myocardial ischemia. This study evaluated sex differences in serotonin and thromboxane A 2 dependent vasoconstriction in human isolated IMA , with the mechanistic role of (1) the endothelium, (2) nitric oxide (NO), (3) prostaglandins, and (4) receptor activity investigated for any observed sex difference. Methods and Results Viable isolated human IMA segments were obtained from 116 patients (44 women [mean age, 66.8±12.2 years] and 72 men [mean age, 66.6±10.4 years]) undergoing coronary artery bypass grafting. Cumulative concentration‐response curves for serotonin and thromboxane A 2 mimetic, U46619, were determined and revealed an increased sensitivity to serotonin but not U46619 in women. This sex difference to serotonin was further assessed by the following: (1) endothelial denudation, (2) endothelial NO synthase inhibition and NO quantification using electron paramagnetic resonance, (3) cyclooxygenase inhibition and prostaglandin metabolite quantification using mass spectrometry, and (4) quantification of receptor activity status. The female hyperreactivity to serotonin was (1) abolished by endothelial denudation; (2) unaffected by NO synthase inhibition, with no difference in electron paramagnetic resonance–assessed NO levels; (3) abolished by cyclooxygenase inhibition (quantification of prostaglandins in IMA revealed a trend towards reduced 6‐keto prostaglandin F1α in female IMA ; P =0.08); and (4) unrelated to receptor activity. Conclusions These data indicate that female IMAs are hyperreactive to serotonin but not U46619, with the former attributable to an endothelium‐dependent cyclooxygenase pathway.

Continuous, polycrystalline ceramic oxide fibers are characterized by having a high alumina content, an ultra-fine or nanoscale microstructure, which provides good strength and flexibility up to 1200°C and above in oxidizing atmospheres. Continuous oxide fibers are produced by the sol/gel process. Fibers are spun into continuous rovings consisting hundreds of individual filaments, which are heat treated to convert them into ceramic. Fibers can be divided into two classes: alumina-silica fibers consisting of a mixture of transitional alumina and amorphous silica and crystalline α-Al2O3–based fibers. Alumina-silica fibers have their largest applications as high-temperature textiles for insulation, while crystalline α-Al2O3fibers have higher elastic modulus and strength that are more commonly used for composite reinforcement. The strength of oxide fibers is controlled by “weakest link” statistics, i.e., fracture occurs at the largest flaw. Minimizing flaw size during processing enables higher strengths. High-temperature fiber properties are controlled by the size and nature of the crystalline and amorphous phases present; fiber manufacturers are working to optimize microstructures to improve high-temperature performance.

DNA minor-groove-binding compounds have limited biological applications, in part due to problems with sequence specificity that cause off-target effects. A model to enhance specificity has been developed with the goal of preparing compounds that bind to two AT sites separated by G•C base pairs. Compounds of interest were probed using thermal melting, circular dichroism, mass spectrometry, biosensor-SPR, and molecular modeling methods. A new minor groove binder that can strongly and specifically recognize a single G•C base pair with flanking AT sequences has been prepared. This multi-site DNA recognition mode offers novel design principles to recognize entirely new DNA motifs.

The design, synthesis, and DNA binding properties of azaHx-PI or p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (5) are described. AzaHx, 2-(p-anisyl)-4-aza-benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, ΔTM, and SPR studies provided evidence that an azaHx/IP pairing, formed from antiparallel stacking of two azaHx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C–G doublet. AzaHx-PI was found to target 5′-ACGCGT-3′, the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity (Keq 4.0 ± 0.2 × 107 M−1).