Design,synthesis and structure–activity relationships of (1H-pyridin-4-ylidene)amines as potential antimalarials

(1H-Pyridin-4-ylidene)amines containing lipophilic side chains at the imine nitrogen atom were prepared as potential clopidol isosteres in the development of antimalarials. Their antiplasmodial activity was evaluated in vitro against the Plasmodium falciparum W2 (chloroquine-resistant) and FCR3 (atovaquone-resistant) strains. The most active of these derivatives, 4m, had an IC₅₀ of 1 μM against W2 and 3 μM against FCR3. Molecular modeling studies suggest that (1H-pyridin-4-ylidene)amines may bind to the ubiquinol oxidation Q_o site of cytochrome bc₁.     

Examining the reliability of the flexor carpi radialis V-wave at different levels of muscle contraction

This study examined the reliability and scaling of the flexor carpi radialis (FCR) V-wave during submaximal and maximal voluntary muscle contractions (MVC). 23 participants were tested on three separate sessions. For each session, participants performed isometric wrist flexions at five contraction levels (20, 40, 60, 80 and 100 %MVC). When the target contraction level was reached, a supramaximal electrical stimulus was applied to the median nerve in order to elicit an FCR V-wave. Across all participants, the FCR V-wave amplitude, normalized to its superimposed M-wave amplitude, increased from 0.030 ± 0.001 to 0.143 ± 0.015 (P < 0.001) as the muscle contraction increased from 20 to 100 %MVC. Contraction level did not influence the reliability of evoking the FCR V-wave, as the V-wave demonstrated both stability and consistency. With the exception of a single day main effect during the 20 %MVC condition, V:M_sup was not different across days or trials (P > 0.05) indicating measurement stability. High reliability co-efficients (0.827–0.913) at each contraction level signified measurement consistency. This study establishes that FCR V-waves can be reliably evoked during both submaximal and maximal muscle contractions and suggests the possibility for FCR V-wave recordings to be used to document neuromuscular adaptations associated with factors such as training or fatigue.     

Effect of corticosterone on growth and welfare of broiler chickens showing long or short tonic immobility

Tonic immobility (TI) test is commonly used to assess fear. Animals showing different TI durations demonstrate distinct behavior and biochemical responses to stress. However, less is known about how TI phenotype affects growth and welfare of domestic fowl. In this study, broiler chickens (Gallus gallus) were classified into short and long TI duration (STI and LTI) phenotypes and treated chronically with vehicle (CON) or corticosterone (CORT). STI broilers demonstrated significantly higher growth rate with higher breast muscle yield (P < 0.05) and liver weight relative to BW tended to be lower (P = 0.053), which was accompanied by higher serum concentration of CORT (P < 0.05) and uric acid (P < 0.01), but lower serum level of T4 (P = 0.01). CORT severely reduced body weight, as well as the relative weight of muscle, bursa of Fabricius and spleen (P < 0.001), but relative liver weight was increased (P < 0.001). CORT-treated chickens had reduced serum CORT, elevated heterophile/lymphocyte ratio, and increased serum levels of total and free T3. STI broilers displayed more preening behavior (P < 0.05), yet CORT elicited more walking behavior (P < 0.05). No difference was observed in the welfare assessment scores between STI and LTI phenotypes under basal situation, while LTI chickens showed significantly increased incidence of pad dermatitis compared to STI under CORT exposure. The results suggest that STI broilers demonstrate better growth performance and higher adaptability to stress compared to LTI chickens.     

New monomeric and dimeric uridinyl derivatives as inhibitors of chitin synthase

This study described the synthesis and in vitro evaluation of eight new derivatives of uridine as antifungal agents and inhibitors of chitin synthase. Dimeric uridinyl derivatives synthesized by us did not exhibit significant activity. One of the studied monomeric derivative, 5′-(N-succinyl)-5′-amino-5′-deoxyuridine methyl ester (compound 7) showed activities against several fungal strains (MIC range 0.06–1.00 mg/mL) and inhibited chitin synthase from Saccharomyces cerevisiae (IC₅₀ = 0.8 mM). Moreover compound 7 exhibited synergistic interaction with caspofungin against Candida albicans (FIC index = 0.28).     

Influence of R genome on the nutritional value of triticale for broiler chicks

The following grain characteristics: protein, arabinoxylan and dietary fibre content, viscosity and water holding capacity of wheat, rye and triticale of different ploidy levels were studied as to their effect on body weight gain (BWG), feed to gain ratio (FCR), apparent metabolizable energy (AME_n), dry matter digestibility (DMD) and apparent protein retention (APR) in young broiler chicks fed isograin and isoprotein diets based on these cereals. Highly significant correlations (p≤0.01) were found between physicochemical and biological quality indicators when all cereals were taken into account. A negative response of chicks to triticale was obtained only when chicks were fed diets containing the tetraploid forms, while the nutrition parameters of chicks fed diets containing the octo- and hexaploid triticale, with rye genome shares of 1 : 3 and 1 : 2, did not differ (p≥0.05) from those fed a wheat diet. Rye diets yielded the lowest BWG, AME_n and DMD and the poorest FCR. The results indicate that as long as the share of the R genome is a minor component of the total triticale genome pool, its antinutritional effect is masked by the wheat genome. The results also indicate that hexaploid triticale can constitute the sole cereal component in the diets of young broiler chicks.     

A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): Purification,primary structure,and inhibitory properties

Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K_a = 1.1 × 10⁹ M^?1 and 2.5 × 10⁹ M^?1, respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K_a = 4.5 × 10⁸ M^?1 and 1.2 × 10¹⁰ M^?1). Weak interaction with human plasmin (K_a = 1.2 × 10⁷ M^?1) was also revealed.     

Antiplasmodial and leishmanicidal activity of biflavonoids from Indian Selaginella bryopteris

A series of eleven biflavonoids containing amentoflavone and hinokiflavone derivatives from the Indian medicinal herb Selaginella bryopteris has been investigated for their antiprotozoal activity using in vitro assays against the K1 strain of Plasmodium falciparum, Leishmania donovani, Trypanosoma brucei rhodesiense and Trypanosoma cruzi. The highest antiprotozoal activity was displayed by 7,4′,7″-tri-O-methylamentoflavone which exhibited an IC₅₀ of 0.26 μM. This compound showed no significant cytotoxicity (IC₅₀ > 150 μM) evaluated using L-6 cells. The strongest activity against Leishmania was detected for 2,3-dihydrohinokiflavone (IC₅₀ = 1.6 μM), whereas for Trypanosoma no significant activity was observed (IC₅₀ > 12.5 μg/mL for the extract). To evaluate the in vivo activity against Plasmodium of the most active compound, trimethylated amentoflavones were obtained by partial synthesis starting from amentoflavone. The synthesized mixture of trimethylated amentoflavones did not show activity in the Plasmodium berghei mouse model against female NMRI mice at 50 mg/kg.     

Antimalarial acridines: Synthesis,in vitro activity against P. falciparum and interaction with hematin

A series of acridine derivatives were synthesised and their in vitro antimalarial activity was evaluated against one chloroquine-susceptible strain (3D7) and three chloroquine-resistant strains (W2, Bre1 and FCR3) of Plasmodium falciparum. Structure–activity relationship showed that two positives charges as well as 6-chloro and 2-methoxy substituents on the acridine ring were required to exert a good antimalarial activity. The best compounds possessing these features inhibited the growth of the chloroquine-susceptible strain with an IC₅₀ ? 0.07 μM, close to that of chloroquine itself, and that of the three chloroquine-resistant strains better than chloroquine with IC₅₀ ? 0.3 μM. These acridine derivatives inhibited the formation of β-hematin, suggesting that, like CQ, they act on the haem crystallization process. Finally, in vitro cytotoxicity was also evaluated upon human KB cells, which showed that one of them 9-(6-ammonioethylamino)-6-chloro-2-methoxyacridinium dichloride 1 displayed a promising antimalarial activity in vitro with a quite good selectivity index versus mammalian cell on the CQ-susceptible strain and promising selectivity on other strains.     

Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana

Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana.     

Design,expression and characterization of the hybrid antimicrobial peptide LHP7, connected by a flexible linker,against Staphylococcus and Streptococcus

The emergence of bacterial resistance to common antibiotics poses a threat to human health and has rekindled an interest in antimicrobial peptides (AMPs). LHP7, a novel hybrid AMP containing 83 amino acid residues was designed on the basis of the LH28 and plectasin. LHP7 was expressed in Pichia pastoris, the total concentration of secreted protein reached 0.906 g/L after 108 h of methanol induction. Its antimicrobial activity was higher than that of the parent AMPs; the minimal inhibitory concentrations (MICs) of LHP7 against Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus suis were 0.091, 0.023 and 0.18 μM, respectively. The antibacterial activity of LHP7 against clinical MRSA isolates (MICs = 0.73–2.91 μM) was enhanced over that of plectasin. The fractional inhibitory concentration (FIC) indicated a synergistic effect between LHP7 and ampicillin against MRSA (FIC = 0.375), and combinations of LHP7 with gentamicin, rifampin or tetracycline provided evidence of additive effects (FIC = 0.625–1.0). LHP7 exhibited a broad range of pH stability and thermostability, and a hemolytic activity of less than 5% below a concentration of 500 μg/mL. It was resistant to pepsin and papain digestion, but sensitive to trypsin digestion. These results suggest that LHP7 might have potential as a broadly applied and clinically useful antimicrobial agent.     

Quantitative evaluation of motor functional recovery process in chronic stroke patients during robot-assisted wrist training

This study was to investigate the motor functional recovery process in chronic stroke during robot-assisted wrist training. Fifteen subjects with chronic upper extremity paresis after stroke attended a 20-session wrist tracking training using an interactive rehabilitation robot. Electromyographic (EMG) parameters, i.e., EMG activation levels of four muscles: biceps brachii (BIC), triceps brachii (TRI, lateral head), flexor carpiradialis (FCR), and extensor carpiradialis (ECR) and their co-contraction indexes (CI) were used to monitor the neuromuscular changes during the training course. The EMG activation levels of the FCR (11.1% of decrease from the initial), BIC (17.1% of decrease from the initial), and ECR (29.4% of decrease from the initial) muscles decreased significantly during the training (P < 0.05). Such decrease was associated with decreased Modified Ashworth Scores for both the wrist and elbow joints (P < 0.05). Significant decrease (P < 0.05) was also found in CIs of muscle pairs, BIC&TRI (21% of decrease from the initial), FCR&BIC (11.3% of decrease from the initial), ECR&BIC (49.3% of decrease from the initial). The decreased CIs related to the BIC muscle were mainly caused by the reduction in the BIC EMG activation level, suggesting a better isolation of the wrist movements from the elbow motions. The decreased CI of ECR& FCR in the later training sessions (P < 0.05) was due to the reduced co-contraction phase of the antagonist muscle pair in the tracking tasks. Significant improvements (P < 0.05) were also found in motor outcomes related to the shoulder/elbow and wrist/hand scores assessed by the Fugl–Meyer assessment before and after the training. According to the evolution of the EMG parameters along the training course, further motor improvements could be obtained by providing more training sessions, since the decreases of the EMG parameters did not reach a steady state before the end of the training. The results in this study provided an objective and quantitative EMG measure to describe the motor recovery process during poststroke robot-assisted wrist for the further understanding on the neuromuscular mechanism associated with the recovery.     

Interactive efficacies of Elephantorrhiza elephantina and Pentanisia prunelloides extracts and isolated compounds against gastrointestinal bacteria

Elephantorrhiza elephantina (Burch.) Skeels (Fabaceae) and Pentanisia prunelloides (Klotzsch ex Eckl. & Zeyh.) Walp. (Rubiaceae) are two medicinal plants used extensively in southern Africa to treat various ailments. Often, decoctions and infusions from these two plants are used in combination specifically for stomach ailments. The antimicrobial activities of the methanol and aqueous extracts of the rhizomes of the two plants, as well as the two active ingredients from the plants [(−)-epicatechin and palmitic acid] have been determined apart and in combination against Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 8739) and Bacillus cereus (ATCC 11778). The minimum inhibitory concentration (MIC) values for the aqueous (0.50–16.00 mg/mL) and methanol (0.20–16.00 mg/mL) extracts independently demonstrated varied efficacies depending on the pathogen of study. When the two plants were combined in 1:1 ratios, synergistic to additive interactions (ΣFIC values 0.19–1.00) were noted. Efficacy for the two major compounds ranged between 0.13–0.63 mg/mL and mainly synergistic interactions were noted against E. faecalis and E. coli. The predominantly synergistic interactions noted between E. elephantina and P. prunelloides and major compounds, when tested in various ratios against these pathogens, provide some validation as to the traditional use of these two plants to treat bacterial gastrointestinal infections.     

S-Arachidonoyl-2-thioglycerol synthesis and use for fluorimetric and colorimetric assays of monoacylglycerol lipase

We report the first synthesis of 2-thioglycerol and S-arachidonoyl-2-thioglycerol (the thioester analog of the endocannabinoid 2-arachidonoylglycerol) in an eight or nine step procedure with a yield of ～25% and establish the use of this substrate for maleimide-based fluorescent and dithiobis(2-nitrobenzoic acid)-based colorimetric assays of human recombinant monoacylglycerol (MAG) lipase (hMAGL) and human brain membrane MAG hydrolase activity. Inhibitor structure–activity relationships observed here for hMAGL and 2-ATG correlate well (r² = 0.93, n = 9) with earlier findings for mouse brain MAG hydrolase with non-thiol substrates.     

Efficacy of biogenic selenium nanoparticles against Leishmania major: In vitro and in vivo studies

Project: This study investigated the in vitro and in vivo effectiveness of biogenic selenium nanoparticles (Se NPs), biosynthesized by Bacillus sp. MSh-1, against Leishmania major (MRHO/IR/75/ER). Procedure: The 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity effects of the biogenic Se NPs against both promastigote and amastigote forms of L. major. In a separate in vivo experiment, we also determined the preventive and therapeutic effects of biogenic Se NPs in BALB/c mice following subcutaneous infected with L. major. Results: The MTT assays showed that the highest toxicity occurred after 72 h against both promastigote and amastigote forms of L. major. The cytotoxicity of Se NPs was higher at all incubation times (24, 48, and 72 h) against the promastigote than the amastigote form (p < 0.05). The 50% inhibitory concentrations (IC₅₀) of the Se NPs were 1.62 ± 0.6 and 4.4 ± 0.6 μg ml^?1 against the promastigote and amastigote forms, respectively, after a 72-h incubation period. Apoptosis assays showed DNA fragmentation in promastigotes treated with Se NPs. In an animal challenge, prophylactic doses of biogenic Se NPs delayed the development of localized cutaneous lesions. Moreover, daily administration of Se NPs (5 or 10 mg kg^?1 day^?1) in similarly infected BALB/c mice that had not received prophylactic doses of Se NPs also abolished the localized lesions after 14 days. Conclusion: Based on these in vitro and in vivo studies, biogenic Se NPs can be considered as a novel therapeutic agent for treatment of the localized lesions typical of cutaneous leishmaniasis.     

Design,synthesis, X-ray studies,and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1′-P2′ ligands

Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1′-P2′ tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (K_i = 13.2 nM, IC₅₀ = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (K_i = 62 pM and 14 pM, respectively) and antiviral activity (IC₅₀ = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.     

Alkaloids from the roots of Stemona javanica (Kunth) Engl. (Stemonaceae) and their anti-malarial,acetylcholinesterase inhibitory and cytotoxic activities

Two new protostemonine-type alkaloids, javastemonine A and B (3 and 4) have been isolated from the root extracts of Stemona javanica together with four known Stemona alkaloids, 13-demethoxy-11(S*),12(R*)-dihydroprotostemonine (1), isoprotostemonine (2), protostemonine and isomaistemonine. The structures and relative configurations of the new alkaloids were determined by spectroscopic analysis. The alkaloids 1 and 2 and protostemonine showed moderated antiplasmodial activities against the Plasmodium falciparum strains, TM4 (IC₅₀ values of 17.7 ± 3.7, 16.8 ± 5.4, 16.0 ± 4.2 μg/mL, respectively) and K1 (IC₅₀ values of 16.8 ± 3.1, 14.1 ± 3.7, 11.9 ± 3.3 μg/mL, respectively). These compounds showed no significant cytotoxicities against KB or Vero cells or acetylcholinesterase inhibitory activities.     

Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum

Trypsin from L. alexandri was purified using only two purification processes: ammonium sulfate precipitation and anion exchange liquid chromatography in DEAE-Sepharose. Trypsin mass was estimated as 24 kDa through SDS-PAGE, which showed only one band in silver staining. The purified enzyme showed an optimum temperature and pH of 50 °C and 9.0, respectively. Stability was well maintained, with high levels of activity at a pH of up to 11.0, including high stability at a temperature of up to 50 °C after 60 min of incubation. The inhibition test demonstrated strong inhibition by PMSF, a serine protease inhibitor, and Kinetic constants km and kcat for BAPNA were 0.517 mM and 5.0 S^?1, respectively. The purified enzyme was also as active as casein, as analyzed by zymography. Therefore, we consider trypsin a promising enzyme for industrial processes, owing to its stability in a wide range of pH and temperature and activity even under immobilization.     

Structural–functional insights of single and multi-domain Capsicum annuum protease inhibitors

Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural–functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. β-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine trypsin indicated three times higher potency of CanPI-7 (K_i ～ 57 μM) than -15 (～184 μM). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity.     

Knipholone cyclooxanthrone and an anthraquinone dimer with antiplasmodial activities from the roots of Kniphofia foliosa

A new phenylanthrone, named knipholone cyclooxanthrone and a dimeric anthraquinone, 10-methoxy-10,7′-(chrysophanol anthrone)-chrysophanol were isolated from the roots of Kniphofia foliosa together with the rare naphthalene glycoside, dianellin. The structures were determined by NMR and mass spectroscopic techniques. The compounds showed antiplasmodial activities against the chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum with 10-methoxy-10,7′-(chrysophanol anthrone)-chrysophanol being the most active with IC₅₀ values of 1.17 ± 0.12 and 4.07 ± 1.54 μg/ml, respectively.