Evaluation of therapeutic effect of Premna herbacea in diabetic rat and isoverbascoside against insulin resistance in L6 muscle cells through bioenergetics and stimulation of JNK and AKT/mTOR signaling cascade

BackgroundPremna herbacea Roxb., a perennial herb is well documented for its therapeutic uses among the traditional health care-givers of Assam, India. Scientific validation on the traditional use of the medicinal plant using modern technology may promote further research in health care.PurposeThis study evaluates the therapeutic potential of methanolic extract of P. herbacea (MEPH) against type 2 diabetes mellitus (T2DM) and its phytochemical(s) in ameliorating insulin resistance (IR), thereby endorsing the plant bioactives as effective anti-hyperglycemic agents.MethodsThe anti-diabetic potential of the plant extract was explored both in L6 muscle cells and high fructose high fat diet (HF-HFD) fed male Sprague Dawley (SD) rats. Bioactivity guided fractionation and isolation procedure yielded Verbascoside and Isoverbascoside (ISOVER) as bioactive and major phytochemicals in P. herbacea. The bioenergetics profile of bioactive ISOVER and its anti-hyperglycemic potential was validated in vitro by XFe24 analyzer, glucose uptake assay and intracellular ROS generation by flourometer, FACS and confocal microscopy. The potential of ISOVER was also checked by screening various protein markers via immunoblotting.ResultsMEPH enhanced glucose uptake in FFA-induced insulin resistant (IR) L6 muscle cells and decreased elevated blood glucose levels in HF-HFD fed rats. Isoverbascoside (ISOVER) was identified as most bioactive phytochemical for the first time from the plant in the Premna genus. ISOVER activated the protein kinase B/AMP-activated protein kinase signaling cascades and enhanced glucose uptake in IR-L6 muscle cells. ISOVER decreased the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) and increased that of mammalian target of rapamycin (mTOR), thereby attenuating IR. However, molecular docking revealed that ISOVER increases insulin sensitivity by targeting the JNK1 kinase as a competitive inhibitor rather than mTOR. These findings were further supported by the bioenergetics profile of ISOVER.ConclusionThis study for the first time depicts the functional properties of ISOVER, derived from Premna herbacea, in ameliorating IR. The phytochemical significantly altered IR with enhanced glucose uptake and inhibition of ROS through JNK-AKT/mTOR signaling which may pave the way for further research in T2DM therapeutics.     

Synthesis and evaluation of novel spiro derivatives for pyrrolopyrimidines as anti-hyperglycemia promising compounds

Pyrrolopyrimidin-4-ylidene-malononitriles IIa–d were prepared as important intermediates for preparation of a new series of spiro-pyrrolopyrimidines. These intermediates undergo cyclisation via reaction with acetylacetone, guanidine hydrochloride or hydrazine hydrate. Elemental and spectroscopic evidences for the structures of these compounds are presented. The final compounds have been monitored for in vivo anti-hyperglycemic activity, compared with Amaryl as standard drug. Among 12 tested compounds, both spiro (pyrano IIIb and pyrazlo Va) derivatives exhibit promising anti-hyperglycemic activity.     

Design,synthesis and α-amylase inhibitory activity of novel chromone derivatives

Quercetin is one of the naturally occurring polyphenol flavonoid predominantly known for antidiabetic activity. In the present study, by considering the structural requirements, twenty two novel chromone derivatives (5–26) as α-amylase inhibitor were designed and subsequently in silico evaluated for drug likeness behavior. Designed compounds were synthesized, characterized by spectral analysis and finally evaluated for the inhibition of α-amylase activity by in vitro assay. Tested compounds exhibited significant to weak activity with IC₅₀ range of 12–125 µM. Among the tested compounds, analogues 5, 8, 12, 13, 15, 17 and 22 exhibited significant human α-amylase inhibitory activity with IC₅₀ values <25 µM, which can be further explored as anti-hyperglycemic agents. Putative binding mode of the significant and least active α-amylase inhibitors with the target enzyme was also explored by the docking studies.     

The Glucose Kinase of Bacillus subtilis

The open reading frame yqgR (now termed glcK), which had been sequenced as part of the genome project, encodes a glucose kinase of Bacillus subtilis. A 1.1-kb DNA fragment containing glcK complemented an Escherichia coli strain deficient in glucose kinase activity. Insertional mutagenesis of glcK resulted in a complete inactivation of glucose kinase activity in crude protein extracts, indicating that B. subtilis contains one major glucose kinase. The glcK gene encodes a 321-residue protein with a molecular mass of 33.5 kDa. The glucose kinase was overexpressed as a fusion protein to a six-His affinity tag and purified to homogeneity. The enzyme had K_m values for ATP and glucose of 0.77 and 0.24 mM, respectively, and a V_max of 93 μmol min⁻¹ mg⁻¹. A B. subtilis strain deficient for glucose kinase grew at the same rate on different carbon sources tested, including disaccharides such as maltose, trehalose, and sucrose.     

Relative Abundance and Plasmodium Infection Rates of Malaria Vectors in and around Jabalpur,a Malaria Endemic Region in Madhya Pradesh State,Central India

BackgroundThis study was undertaken in two Primary Health Centers (PHCs) of malaria endemic district Jabalpur in Madhya Pradesh (Central India).MethodsIn this study we had investigated the relative frequencies of the different anopheline species collected within the study areas by using indoor resting catches, CDC light trap and human landing methods. Sibling species of malaria vectors were identified by cytogenetic and molecular techniques. The role of each vector and its sibling species in the transmission of the different Plasmodium species was ascertained by using sporozoite ELISA.ResultsA total of 52,857 specimens comprising of 17 anopheline species were collected by three different methods (39,964 by indoor resting collections, 1059 by human landing and 11,834 by CDC light trap). Anopheles culicifacies was most predominant species in all collections (55, 71 and 32% in indoor resting, human landing and light trap collections respectively) followed by An. subpictus and An. annularis. All five sibling species of An. culicifacies viz. species A, B, C, D and E were found while only species T and S of An. fluviatilis were collected. The overall sporozoite rate in An. culicifacies and An. fluviatilis were 0.42% (0.25% for P. falciparum and 0.17% for P. vivax) and 0.90% (0.45% for P. falciparum and 0.45% for P. vivax) respectively. An. culicifacies and An. fluviatilis were found harbouring both P. vivax variants VK-210 and VK-247, and P. falciparum. An. culicifacies sibling species C and D were incriminated as vectors during most part of the year while sibling species T of An. fluviatilis was identified as potential vector in monsoon and post monsoon season.ConclusionsAn. culicifacies species C (59%) was the most abundant species followed by An. culicifacies D (24%), B (8.7%), E (6.7%) and A (1.5%). Among An. fluviatilis sibling species, species T was common (99%) and only few specimens of S were found. Our study provides crucial information on the prevalence of An. culicifacies and An. fluviatilis sibling species and their potential in malaria transmission which will assist in developing strategic control measures against these vectors.     

Structure,dynamics, and biochemical characterization of ADF/cofilin Twinstar from Drosophila melanogaster

BackgroundTwinstar is an ADF/cofilin family protein, which is expressed by the tsr gene in Drosophila melanogaster. Twinstar is one of the main regulators of actin cytoskeleton remodelling and is essential for vital cellular processes like cytokinesis and endocytosis.MethodsWe have characterized the structure and dynamics of Twinstar by solution NMR spectroscopy, the interaction of Twinstar with rabbit muscle actin by ITC, and biochemical activities of Twinstar through different biochemical assays using fluorescence spectroscopy and ultra-centrifugation.ResultsThe solution structure of Twinstar shows characteristic ADF-H fold with well-formed G/F-site and F-site for interaction with actin. The structure possesses an extended F-loop, which is rigid at the base, but flexible towards its apical region. Twinstar shares similar dynamics for the G/F-site with C. elegans homologs, UNC-60A and UNC-60B. However, the dynamics of its F-loop are different from its C. elegans homologs. Twinstar shows strong affinity for ADP-G-Actin and ATP-G-Actin with K_ds of ~7.6 nM and ~0.4 μM, respectively. It shows mild F-actin depolymerizing activity and stable interaction with F-actin with a K_d of ~5.0 μM. It inhibits the rate of the nucleotide exchange in a dose dependent manner.ConclusionOn the basis of structure, dynamics, and biochemical activity, Twinstar can be taken to execute its biochemical role by facilitating directional growth and maintenance of length of actin filaments.General significanceThis study characterizes the structure, backbone dynamics, and biochemical activities of Twinstar of Drosophila, which provides an insight into the regulation of actin dynamics in the member of phylum insecta.     

The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans

Several families of crystal proteins from Bacillus thuringiensis exhibit nematicidal activity. Cry5B protein, a pore-forming toxin, has been intensively studied yielding many insights into the mode of action of crystal protein at molecular level and pathogenesis of pore-forming toxins. However, little attention was paid to Cry6A, another representative nematicidal crystal protein. Cry6A shares very low homology with Cry5B at amino acid sequence and probably acts in a distinct pathway from Cry5B and even the other main commercial crystal proteins. In the current study, we comprehensively investigated the nematicidal properties of Cry6Aa2 against the free-living soil nematode Caenorhabditis elegans and examined the physical response of C. elegans to Cry6Aa2 attack. Our results indicate that Cry6Aa2 exhibits high lethal activity to C. elegans and could cause detrimental effects on C. elegans, including obviously suppressed growth, decreased brood size, and even abnormal motility. Meanwhile, our study additionally shows that C. elegans could defend against the Cry6Aa2 toxin harmful threat through behavioral defense responses, such as reduced oral uptake and physical avoidance. In general, this study suggests that Cry6Aa2 possesses diverse nematicidal properties, which strongly indicates that Cry6Aa2 is a promising potential candidate of nematicidal agent. Moreover, this study highlights the importance of behavioral responses in defense of C. elegans for survival and demonstrates the key role of crystal protein in the interaction of B. thuringiensis–C. elegans. These findings could shed light on understanding the interaction of C. elegans with B. thuringiensis and provide a perfect model to study the role of pathogenic factor in the interaction of pathogen–host.     

Diurnal Glucose Profiles Using Continuous Glucose Monitoring to Identify the Glucose-Lowering Characteristics of Colesevelam HCL (WELCHOL)

ObjectiveThe study's purpose was to identify the antihyperglycemic affects of colesevelam-HCl (C-HCl) by characterizing the diurnal and postprandial glucose patterns in type 2 diabetic subjects treated concomitantly with metformin, sulfonylurea, or a combination of metformin/ sulfonylurea. A secondary aim was to determine whether C-HCl significantly increased the risk of hypoglycemia.MethodsA prospective, randomized, double-blind, placebo-controlled, crossover study employing continuous glucose monitoring (CGM) with ambulatory glucose profile (AGP) analysis was undertaken. Fifteen males and 6 females, age 60 ± 8 years, treated with metformin (n = 8), sulfonylurea (n = 2), or combination (n = 11) participated.ResultsTreatment with C-HCl led to reductions in glycated hemoglobin (HbAlc) (7.5 ± 0.3 to 7.0 ± 0.4% P<.0001), LDL (90.9 ± 18.6 to 68.9 ± 15.2 mg/dL, P<.0007) and total cholesterol (169.2 ± 24.4 to 147.8 ± 21.5 mg/dL, P<.001). Significantly lower normalized diurnal (21 mg/dL/hour, P = .0006), nocturnal (19 mg/dL/hour, P = .0005), and daytime (22 mg/dL/hour, P = .0008) glucose exposure was detected immediately upon C-HCl administration. Additionally, there was a significant (P<.004) decline in postprandial glucose excursions (averaging 15% or -36 mg/dL/hour) pronounced at dinner following C-HCl administration. There was a nonsignificant increase in the incidence of hypoglycemia (0.4-1%), with no difference due to antihyperglycemic medications.ConclusionsAGP analysis of CGM visually and quantitatively showed immediate and midterm impacts of C-HCl on basal and postprandial glucose patterns. This suggests a multifactorial glucose-lowering mechanism for C-HCl affecting both meal-related and basal glucose levels. (Endocr Pract. 2013;19:275-283)     

Comparison of sand fly trapping approaches for vector surveillance of Leishmania and Bartonella species in ecologically distinct,endemic regions of Peru

BackgroundIn Peru, the information regarding sand fly vectors of leishmaniasis and bartonellosis in the Amazon region is limited. In this study, we carried out sand fly collections in Peruvian lowland and highland jungle areas using different trap type configurations and screened them for Leishmania and Bartonella DNA.Methodology/Principal findingsPhlebotomine sand flies were collected in Peruvian Amazon jungle and inter Andean regions using CDC light trap, UV and color LED traps, Mosquito Magnet trap, BG Sentinel trap, and a Shannon trap placed outside the houses. Leishmania spp. screening was performed by kDNA PCR and confirmed by a nested cytochrome B gene (cytB) PCR. Bartonella spp. screening was performed by ITS PCR and confirmed by citrate synthase gene (gltA). The PCR amplicons were sequenced to identify Leishmania and Bartonella species.UV and Blue LED traps collected the highest average number of sand flies per hour in low jungle; UV, Mosquito Magnet and Shannon traps in high jungle; and Mosquito Magnet in inter Andean region. Leishmania guyanensis in Lutzomyia carrerai carrerai and L. naiffi in Lu. hirsuta hirsuta were identified based on cytB sequencing. Bartonella spp. related to Bartonella bacilliformis in Lu. whitmani, Lu. nevesi, Lu. hirsuta hirsuta and Lu. sherlocki, and a Bartonella sp. related to Candidatus B. rondoniensis in Lu. nevesi and Lu. maranonensis were identified based on gltA gene sequencing.Conclusions/SignificanceUV, Blue LED, Mosquito Magnet and Shannon traps were more efficient than the BG-Sentinel, Green, and Red LED traps. This is the first report of L. naiffi and of two genotypes of Bartonella spp. related to B. bacilliformis and Candidatus B. rondoniensis infecting sand fly species from the Amazon region in Peru.     

Design,synthesis and biological evaluation of novel pyrimidinedione derivatives as DPP-4 inhibitors

A series of novel pyrimidinedione derivatives were designed and evaluated for in vitro dipeptidyl peptidase-4 (DPP-4) inhibitory activity and in vivo anti-hyperglycemic efficacy. Among them, the representative compounds 11, 15 and 16 showed excellent inhibitory activity of DPP-4 with IC₅₀ values of 64.47?nM, 188.7?nM and 65.36?nM, respectively. Further studies revealed that compound 11 was potent in vivo hypoglycemic effect. The structure–activity relationships of these pyrimidinedione derivatives had been discussed, which would be useful for developing novel DPP-4 inhibitors as treating type 2 diabetes.     

In-vivo hyperglycemic,antioxidant, histopathological changes,and simultaneous measurement of kaempferol verified by high-performance thin layer chromatography of Setaria italica in streptozotocin -induced diabetic rats

BackgroundSetaria italica (common name- foxtail, kangni) is one of the major food crops which is prominently cultivated in southern regions of India and in certain regions of Uttar Pradesh. Besides the crop’s consumption as a general source of carbohydrate rich cereal, the seeds of the crop are comprised of more fiber. So, it is recommended to add in the dietary supplementation of the diabetic people across the country.ObjectiveIn this paper, it intends to investigate the antidiabetic activity and antioxidant activity of S. italica (foxtail millet) seeds in diabetic rats.MethodsThe six genotypes of foxtail millets (S. italica) namely Kangni-1, Kangni-4, Kangni-5, Kangni-6, Kangni-7 & Kangni-10 respectively were subjected to in vitro investigations via. comprehensive metabolic panel (CMP) involving blood glucose study, Kidney & Liver function test, and antioxidant study (Catalase test; Glutathione S-transferase (GST); Superoxide Dismutase (SOD); glutathione (GSH); hiobarbituric acid reactive substances (TBARS) & Glutathione peroxidase (GPx) and were performed in vivo animal investigations in Wistar rats. The STZ induced diabetic rats were fed with doses of different S. italica seed aqueous extract to evaluate its anti-hyperglycemic activity by oral administration of SISAE. Further, it was compared with Glibenclamide which acts as one of the standard oral hypoglycemic agents.ResultsFrom achieved outcomes, a significant fall of blood glucose level (70%) produced 300 mg SISAE/kg b.w. after 6 h of extract administration. However, no change could be produced by these doses of the SISAE in normal rats’ blood glucose levels. A significant fall in glucose level along with significant glycemic control by lower HbA1c levels was observed in diabetic treated rats after 3 weeks of treatment with 300 mg of SISAE/kg b.w./day when comparing to untreated diabetic rats. Among these five genotypes of S. italica, the differences in the glycemic index were found. a significant fall could be found in blood glucose levels of Wistar rats, when every experimental rat was incorporating with the extract of different genotypes of Setaria italica L. Beauv than the rats treated with Glibenclamide in every 7 days of interval. The level of catalase, SOD, GST, GPx, GSH and TBARS showed variation while the rats were fed with the extract of S. italica in the liver test of rats. In kidney function test, the result shows that there is significant relationship between foxtail extract and kidney function of STZ induced diabetes rats. They show the change in their serum creatinine level, serum urea and serum uric acid.ConclusionThe result obtained from the study shows that the extract of S. italica seeds is capable for the hypolipidemic and antihyperglycemic activities, thereby, they serve as one of the good sources for herbal medicinal items.     

Synthesis and molecular docking study of some 3,4-dihydrothieno[2,3-d]pyrimidine derivatives as potential antimicrobial agents

In continuation of our research program aiming at developing new potent antimicrobial agents, new series of substituted 3,4-dihydrothieno[2,3-d]pyrimidines was synthesized. The newly synthesized compounds were preliminary tested for their in vitro activity against six bacterial and three fungal strains using the agar diffusion technique. The results revealed that compounds 7, 8a, 10b, 10d and 11b exhibited half the potency of levofloxacine against the Gram-negative bacterium, Pseudomonas aeruginosa, while compounds 5a, 8b, 10c and 12 displayed half the potency of levofloxacine against Proteus Vulgaris. Whereas, compounds 7, 10b, 10d and 11b showed half the activity of ampicillin against the Gram-positive bacterium, B. subtilis. Most of the compounds showed high antifungal potency. Compounds 3, 6, 7, 9b, 10a, 11a, 11b, 15 and 16 exhibited double the potency of clotrimazole against A. fumigatus. While compounds 3, 4, 5a, 5b, 9b, 10a, 10b, 10c, 13, 15, 16 and 18 displayed double the activity of clotrimazole against R. oryazae. Molecular docking studies of the active compounds with the active site of the B. anthracis DHPS, showed good scoring for various interactions with the active site of the enzyme compared to the co-crystallized ligand.     

In vitro antioxidant,antimicrobial and antiproliferative studies of four different extracts of Orthosiphon stamineus,Gynura procumbens and Ficus deltoidea

BackgroundMedicinal plants are important source of drugs with pharmacological activities. Therefore, there is always rising demands to discover more therapeutic agents from various species. Orthosiphon stamineus, Gynura procumbens and Ficus deltoidea are high valued medicinal plants of Malaysia contain rich source of phenolic and flavonoid compounds. The aims of the present study were to evaluate anti-oxidant, antimicrobial and anti-proliferative effects on A549, HePG2 and MCF7 cell lines of four different extracts of Orthosiphon stamineus, Gynura procumbens and Ficus deltoidea.MethodologyThe leaves of all selected plants were extracted with methanol, chloroform, ethyl acetate and butanol separately with simple cold maceration. Antioxidant activity of all crude extracts were quantitatively measured against DPPH and Ferric Reducing Assay. Antimicrobial evaluation was done by Microdilution and MTT assay and antipoliferative activity of all extracts of selected plant were evaluated against A549, HePG2 and MCF7 cell lines.ResultsResults showed that methanol extract exhibited highest percentage free radical scavenging activity of almost all extracts of selected plants. Antimicrobials results showed chloroform and methanol extracts of O. stamineus extract were the two most active extracts against resistant MRSA but not S. aureus. Only methanol extract of G. procumbens showed antimicrobial activity against the tested pathogens. Chloroform and methanol extracts of F. deltoidea elicited antimicrobial activity against S. aureus but not MRSA. Antiproliferative activity against three tested cell lines results showed that ethyl acetate extract of O. stamineus showed good effect whereas methanol extract of F. deltoidea and G. procumbens exhibited good antiproliferative activity.ConclusionsThe results of the present investigation demonstrated significant variations in the antioxidant, antimicrobial and antiproliferative effects of different solvent extracts. These data could be helpful in isolation of pure potent compounds with good biological activities from the extracts of plants.     

Nitrochalcones: Potential in vivo insulin secretagogues

In this study, the in vivo and in vitro anti-hyperglycemic activity of chalcone derivatives of 3,4-methylenedioxy, with a substituent electron-acceptor nitro group in the A or B ring, was investigated. As expected, the second generation sulfonylurea glipizide stimulated insulin secretion and reduced glycemia over the study period. Also, it was demonstrated for the first time that chalcones are able to increase insulin secretion and this event was coincident with serum glucose-lowering in the oral glucose tolerance test. Additionally, the chalcones studied had a similar effect on insulin secretion and serum glucose-lowering as glipizide. The effect of chalcones in terms of inducing insulin secretion was greater than that of glipizide after 30 min. Moreover, chalcones were not able to stimulate glucose uptake in soleus muscle, either in the presence of insulin or in the absence of this hormone. In addition, the oral treatment with chalcones did not alter glycemia in diabetic rats. These reports indicate that the effect of chalcones on serum glucose-lowering in hyperglycemic-normal rats is mainly a consequence of insulin secretion, highlighting these chalcones as novel compounds with strong anti-hyperglycemic properties.     

A Conformationally Constrained Cyclic Acyldepsipeptide Is Highly Effective in Mice Infected with Methicillin-Susceptible and -Resistant Staphylococcus aureus

BackgroundCyclic acyldepsipeptides (ADEPs) are a novel class of antibacterial agents, some of which (e.g., ADEP 4) are highly active against Gram-positive bacteria. The focus of these in vivo studies is ADEP B315, a rationally designed compound that has the most potent in vitro activity of any ADEP analog reported to date.MethodsIn vivo efficacy experiments were performed using lethal intraperitoneal mice infection models with a methicillin-sensitive S. aureus (MSSA) and a methicillin-resistant (MRSA) strain. The infected mice were treated with ADEP B315, a des-methyl analog of ADEP 4, vancomycin, or the vehicle used for the ADEPs and their survival was assessed daily. A subset of MSSA-infected mice was sacrificed soon after inoculation and the bacterial burden was measured in their livers and spleens. The toxicity of ADEP B315 was assessed in viability assays using human whole blood cultures.ResultsIn the MSSA experiments, all mice treated with the vehicle succumbed to the infection within 24 hours. All tested compounds were effective in prolonging survival of infected mice (p<0.001). Mice treated with ADEP B315 had a 39% survival rate by 10 days compared to 7% survival in mice treated with a des-methyl ADEP 4 analog (p = 0.017). Survival of the infected mice treated with ADEP B315 was comparable to those treated with vanocmycin (p = 0.12) at the same dose. Further, bacterial burden in the liver and spleen was significantly lower in mice treated with ADEP B315 compared to controls. In the MRSA experiments, ADEP B315 was able to significantly prolong survival compared to mice treated with either the vehicle (p = 0.001) or vancomycin (p = 0.007). ADEP B315 exhibited no significant toxicity in human whole blood cultures at concentrations up to 25 μg/ml.ConclusionsADEP B315 is safe and can cure mice that have lethal infections of methicillin-sensitive and -resistant strains of S. aureus.     

Burkholderia species in human infections in Mexico: Identification of B. cepacia,B. contaminans,B. multivorans,B. vietnamiensis,B. pseudomallei and a new Burkholderia species

BackgroundBurkholderia sensu stricto is comprised mainly of opportunistic pathogens. This group is widely distributed in the environment but is especially important in clinical settings. In Mexico, few species have been correctly identified among patients, most often B. cepacia is described.Methodology/Principal findingsIn this study, approximately 90 strains identified as B. cepacia with the VITEK2 system were isolated from two medical centers in Mexico City and analyzed by MLSA, BOX-PCR and genome analysis. The initial identification of B. cepacia was confirmed for many strains, but B. contaminans, B. multivorans and B. vietnamiensis were also identified among clinical strains for the first time in hospitals in Mexico. Additionally, the presence of B. pseudomallei was confirmed, and a novel species within the B. cepacia complex was documented. Several strains misidentified as B. cepacia actually belong to the genera Pseudomonas, Stenotrophomonas and Providencia.Conclusions/SignificanceThe presence of different Burkholderia species in Mexico was confirmed. Correct identification of Burkholderia species is important to provide accurate treatment for immunosuppressed patients.     

Molecular identification of Botrytis cinerea,Botrytis paeoniae and Botrytis pseudocinerea associated with gray mould disease in peonies (Paeonia lactiflora Pall.) in Southern Chile

BackgroundIn Chile, the peony is the most important ornamental flower exported from the country. Gray mould is a phytopathological problem of this crop. This disease is caused by Botrytis cinerea and Botrytis paeoniae.AimsWe carried out the first survey of Botrytis species associated with peony gray mould in Southern Chile to estimate the diversity of these pathogens.MethodsDiseased peony leaves were collected from seven locations in Southern Chile covering a distance of 300 km. The Botrytis isolates obtained were studied by morphological and molecular methods. Finally, a PCR assay using primers based on the necrosis and ethylene-inducing protein gene (nep1) was used to specifically identify B. paeoniae.ResultsSeventeen isolates belonging to Botrytis genus were obtained, and all of them were pathogenic to peonies when inoculated in plants grown in a greenhouse. Morphological analyses showed that four isolates shared common characteristics, which distinguish them from the rest. Homology and phylogenetic analysis of G3PDH, as well as determination of the Bc-hch allele, allowed us to identify 12 isolates as B. cinerea, 4 as B. paeoniae and one isolate as Botrytis pseudocinerea. The PCR assay was found to be specific to B. paeoniae, amplifying a single band of 470 bp.ConclusionsThree Botrytis species involved in peony gray mould disease are present in Chile. This is the first time that both B. paeoniae and B. pseudocinerea have been reported to be present in the country and also that they affect peonies. Finally, to our knowledge, the PCR based method herein described is the first of its kind to be used to identify B. paeoniae.     

Diurnal Glucose Patterns of Exenatide Once Weekly: A 1-Year Study Using Continuous Glucose Monitoring with Ambulatory Glucose Profile Analysis

ObjectiveTo use continuous glucose monitoring (CGM) to characterize diurnal glucose patterns produced by a novel formulation of exenatide consisting of biodegradable polymeric microspheres that entrap exenatide and provide extended release enabling once-weekly administration.MethodsWe performed a subgroup analysis of patients with type 2 diabetes who participated in a multicenter trial (DURATION-1: Effects of Exenatide Long- Acting Release on Glucose Control and Safety in Subjects With Type 2 Diabetes Mellitus) comparing once-weekly with twice-daily formulations of exenatide. We are the only center to use CGM with ambulatory glucose profile (AGP) analysis to characterize glucose exposure, variability, and stability in participants assigned to exenatide once weekly.ResultsSeven of the 303 patients in the larger study population were included in the subgroup analysis. Mean age (57.6 ± 7 years), weight (102 ± 17 kg), body mass index (34 ± 3 kg/m²), and duration of diabetes (5 ± 2 years) were comparable to characteristics of the larger study population. At 30 weeks and 52 weeks, participants treated with exenatide once weekly had a mean reduction in hemoglobin A_1c level of 1.3 ± 0.3% and 1.0 ± 0.3%, respectively (P < .05). CGM analysis revealed a significant (P < .01) decrease in diurnal glucose exposure for 4 participants during nocturnal and daytime periods. Excess glucose exposure (compared with reference values) decreased in 6 of 7 participants, as did glucose variability. Glucose stability improved in 5 participants. The percentage of glucose values less than 70 mg/dL initially increased during the first half of the study then decreased to baseline levels by study end.ConclusionsIndividual glucose profiles revealed that changes in hemoglobin A_1c did not consistently parallel alterations in glucose exposure, variability, and stability. AGPs provided a visual representation of improved glucose responses to exenatide once weekly. (Endocr Pract. 2009;15:326-334)     

Inhibitors of the Candida albicans Major Facilitator Superfamily Transporter Mdr1p Responsible for Fluconazole Resistance

ObjectiveTo identify a novel class of inhibitors of fungal transporters involved in drug resistance.MethodsA series of structurally-related low molecular mass compounds was synthesized using combinatorial chemistry of a cyclobutene-dione (squarile) core. These compounds were screened for their inhibition of plasma membrane Major Facilitator Superfamily (MFS) and ATP-binding cassette (ABC) transporters responsible for efflux pump-mediated drug resistance in the fungal pathogen Candida albicans. Strains of Saccharomyces cerevisiae that specifically overexpress the MFS pump CaMdr1p or the ABC transporter CaCdr1p were used in primary screens and counterscreens, respectively, and to detect inhibition of glucose-dependent Nile Red efflux. Efflux pump inhibition, activity as pump substrates and antifungal activity against yeast and clinical isolates expressing efflux pumps were determined using agarose diffusion susceptibility assays and checkerboard liquid chemosensitization assays with fluconazole.ResultsThe screen identified five structurally-related compounds which inhibited CaMdr1p. Two compounds, A and B, specifically chemosensitized AD/CaMDR1 to FLC in a pH-dependent fashion and acted synergistically with FLC in checkerboard liquid MIC assays but compound B had limited solubility. Compound A chemosensitized to FLC the azole-resistant C. albicans strain FR2, which over-expresses CaMdr1p, inhibited Nile Red efflux mediated by CaMdr1p but not CaCdr1p and was not toxic to cultured human cells. A minor growth-inhibitory effect of B on AD/CaMDR1, but not on AD/CaCDR1 and AD/CaCDR2, indicated that compound B may be a substrate of these transporters. The related compound F was found to have antifungal activity against the three pump over-expressing strains used in the study.ConclusionsCompound A is a ‘first in class’ small molecule inhibitor of MFS efflux pump CaMdr1p.