target site mechanism of resistance

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Target-site resistance mutations (kdr and RDL), but not metabolic resistance, negatively impact male mating competiveness in the malaria vector Anopheles gambiae

Target-site resistance mutations (kdr and RDL), but not metabolic resistance, negatively impact male mating competiveness in the malaria vector Anopheles gambiae

Metabolic resistance has limited fitness cost on mating success In contrast to the target site resistance mechanism, metabolic resistance was not found to significantly influence the mating competitiveness of male An. coluzzii mosquitoes. First, most of the detoxification genes associated with resistance to insecticides in VK such as CYP6P3, CYP6Z2, CYP6M2 and aldehyde oxidase (Kwiatkowska et al., 2013) were not differentially expressed between the mated and unmated males suggesting that overexpression of these genes does not impact mating success. However, a P450, CYP6P2, overexpressed in resistant mosquitoes was found to be downregulated in males that mated suggesting that, although not common, some metabolic resistance genes could still be associated with a negative impact on mating competiveness. Second, the fact that 16 out of 19 resistance genes upregulated in resistant mosquitoes in VK and also differentially expressed between mated and unmated mosquitoes were rather upregulated in mated males further suggests that overexpression of detoxification genes does not provide a fitness disadvantage to male An. coluzzii mosquitoes, at least in terms of mating success. This is also supported by the enrichment of GO terms associated with peroxidase and cytochrome P450s in mated mosquitoes. The lack of fitness cost associated with metabolic resistance in this study could be explained by the fact that metabolic resistance genes belong to large gene families with broad catalytic activities and therefore are unlikely to be adversely affected by mutations in the same way as specific target site mutations affect genes such as the sodium channel, with vital functions and the need for a highly conserved genomic sequence. Therefore, metabolic resistance, notably through over-expression as found for the VK population, can confer a high level of protection against insecticides without significant fitness cost to the resistant mosquitoes in relation to mating. However, it has previously been reported that other metabolic resistance mechanisms such as the overproduction of carboxylesterases could confer a significant fitness cost as observed in resistant C. pipiens, which shows a reduced locomotive performance than susceptible ones. It was suggested that such reduced performance was caused by a resource depletion linked to the overproduction of carboxylesterases (Berticat et al., 2004). However, such overproduction of a specific gene was not observed in VK as the fold change of detoxification genes was not too high, with the highest being for the P450 CYP6Z2 genes at FC20.5.

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Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

The intrinsic mannose patch is targeted by the so-called “man- nose patch-dependent” antibodies, which include PGT121 to -124, 10-1074, PGT125 to -128, PGT130 and -131, PGT135 to -137, and 2G12 (14–16, 26–29). These antibodies display remark- able potencies against a diverse panel of HIV-1 strains, although their breadth varies both between and within families (2, 30). PGT135 was found to neutralize 33% of viruses from a 162-cross- clade-pseudovirus panel. This neutralization is equivalent to the breadth of b12, which has a protein-based epitope at the CD4 binding site, but is lower than those of other Asn332-dependent bnAbs, such as PGT128 and PGT121, which neutralized 72% and 70% of the panel, respectively (2). This lower breadth of neutral- ization has been attributed to the limited prevalence of the larger number of critical contact residues (Asn332, Asn392, and His330) across different isolates (15) compared to PGT121 and PGT128. In addition to these properties, inspection of neutralization pro- files reveals that, despite containing the required target residues, for some strains of HIV-1, neutralization is incomplete, with pla- teaus that do not reach 100% (15). A crystal structure of a PGT135 Fab domain in complex with the gp120 core revealed that the majority of the interactions were mediated through contact with the glycans at the Asn332, Asn392, and Asn386 sites, with 1,010 Å 2

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Insecticide resistance and target site mutations (G119S ace 1 and L1014F kdr) of Culex pipiens in Morocco

Insecticide resistance and target site mutations (G119S ace 1 and L1014F kdr) of Culex pipiens in Morocco

There are different types of insecticide resistance mechanisms in mosquitoes, but the two main ones are (i) the metabolic resistance, grouping three major fam- ilies of enzymes involved, glutathione S-transferase (GST), multifunctional monooxygenases (MFOs) and carboxyl-esterase (COE), and (ii) the target site modifi- cation, which is due to point mutations in the target of insecticides, thus limiting the binding of the neurotoxin products. Several insecticide target modifications have been described: the γ-amino butyric acid (GABA Receptors) encoded by Rdl gene, the synaptic acethyl- cholinesterase (AChE1) encoded by the ace-1 gene and the voltage-dependent sodium channel encoded by the kdr gene [9, 10]. The knockdown resistance kdr gene is the major mechanism responsible for resistance to DDT and PYR, reducing the sensitivity of the receptors to these products in the voltage-gated sodium channel (VGSC) across the neural axon [8, 11]. This resistance is due to the mutations of kdr gene. Many mutations have been reported, but the most common known to be asso- ciated with knockdown resistance in mosquitoes, includ- ing Cx. pipiens, are L1014F by the substitution of a leucine (TTA) by phenylalanine (TTT) and L1014S by the leucine (TTA) to serine (TCA) substitution at codon 1014 [12 – 14], while the L1014C mutation by the substi- tution of a leucine (TTA) by cysteine (TGT) has only been reported for Cx. pipiens molestus from China [15].

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Molecular evidence for historical presence of knock down resistance in Anopheles albimanus, a key malaria vector in Latin America

Molecular evidence for historical presence of knock down resistance in Anopheles albimanus, a key malaria vector in Latin America

Resistance to pyrethroid insecticides in malaria vectors can be primarily mediated by either metabolic mecha- nisms or target site insensitivity, such as mutations on the voltage-gated sodium channel (VGSC) gene [3,18]. Despite reports of pyrethroid resistance throughout the region, none of these mechanisms have been well-described at the molecular level for malaria vectors in Latin America [19]. Previous studies using biochemical assays and bioassays with synergists on pyrethroid resistant An. albimanus from Guatemala and Mexico suggest that an increase in the activity levels of esterases and multi-function oxidases are at least partially responsible for the resistance detected in these populations [20-24]. Elevated oxidase activity has been associated with cross-resistance to pyrethroids and DDT in An. albimanus [23]. One previous study carried out on An. albimanus from Mexico suggested that a target-site mechanism may be involved in cross-resistance between pyrethroids and DDT [25]. Knock-down resist- ance (kdr) is a target-site mechanism reported in other anopheline species that results in cross-resistance to both pyrethroids and DDT [26,27]. In anophelines, kdr is linked to single nucleotide polymorphisms on transmembrane segment 6 of domain II of the VGSC gene. The mutations previously reported for anophelines occur on codon 1014, resulting in an amino acid change of leucine to phenyl- alanine, serine or cysteine [28-34]. To date, similar muta- tions have not been described in An. albimanus.

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Alterations in life-history associated with non-target-site herbicide resistance in Alopecurus myosuroides

Alterations in life-history associated with non-target-site herbicide resistance in Alopecurus myosuroides

Plant evolutionary theory predicts that adaptations conveying resistance to an environmental stress can result in an ecological fitness cost in the absence of the stress, relative to individuals without the adaptation (Coustau et al., 2000; Heil, 2002). Such fitness costs can be allocation based, whereby limited plant resources are constitutively allocated to defense instead of to growth or reproduction (e.g., Herms and Mattson, 1992; Strauss et al., 2002). Alternatively, resistance mutations may directly affect enzyme-mediated reactions, e.g., via reduced catalytic capacity or substrate affinity, resulting in a subsequent fitness cost (Vila-Aiub et al., 2015). Some empirical evidence in support of this has been observed, for example costs of both constitutive and induced defense against herbivory have been documented (Strauss et al., 2002). Nevertheless, despite a sound theoretical background, such costs have remained difficult to conclusively demonstrate across taxa and disciplines (e.g., Laine, 2016). The rapid evolution of resistance to xenobiotics (e.g., fungicides, insecticides, herbicides) provides an exemplary model system with which to study such fitness trade-offs (Coustau et al., 2000; Vila-Aiub et al., 2011). In weeds, resistance to herbicides can evolve via mutations which alter expression of the herbicide target enzyme or reduce herbicide-target binding (target site resistance, TSR), or via changes in the rates of herbicide absorption, translocation, degradation or sequestration (non-target-site resistance, NTSR), (Powles and Yu, 2010). Within NTSR, enhanced metabolism of the herbicide molecule (EMR) is now widely reported as an important resistance mechanism (Yuan et al., 2007; Délye, 2013; Yu and Powles, 2014; Scarabel et al., 2015), and the extent to which this mechanism affects plant fitness is now being increasingly considered (Vila-Aiub et al., 2005a, 2009a; Keshtkar et al., 2017a,b).

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Non‐target site SDHI resistance is present as standing genetic variation in field populations of Zymoseptoria tritici

Non‐target site SDHI resistance is present as standing genetic variation in field populations of Zymoseptoria tritici

Our aim of this study is: (i) to confirm a cross-resistance relation- ship between SDHIs with similar chemical structures, (ii) to check the distribution of resistance against fluopyram and isofetamid in a population collected at different locations and over time, and (iii) to investigate the resistance mechanism. Here, we report fur- ther studies on the detection and characterization of fluopyram- and isofetamid-resistant Z. tritici field strains isolated from differ- ent countries. Isofetamid is not commercialized as a cereal diseases control agent, but fluopyram has just been introduced in the UK in a mixture with bixafen and prothioconazole to enhance and obtain a wider spectrum of disease control. We observed a pos- itive cross-resistance relationship between fluopyram and isofe- tamid. Sequencing analysis of the SDHI binding pocket (SdhB, SdhC and SdhD) and mitochondrial Sdh enzyme activity assays revealed that the inhibitory effects of both fluopyram and isofetamid were severely impaired in the absence of any target-site alterations in resistant strains. Further studies are needed to elucidate the under- lying resistance mechanism(s) in these strains.

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Acetylcholinesterase (Ace 1) target site mutation 119S is strongly diagnostic of carbamate and organophosphate resistance in Anopheles gambiae s s  and Anopheles coluzzii across southern Ghana

Acetylcholinesterase (Ace 1) target site mutation 119S is strongly diagnostic of carbamate and organophosphate resistance in Anopheles gambiae s s and Anopheles coluzzii across southern Ghana

Interestingly however, species was not a significant pre- dictor of resistance when Ace-1 119S genotype was in- cluded in models, suggesting that the primary cause of the difference relates to the target site polymorphism rather than wider genomic background in these sam- ples. It has been suggested that the difference in 119S mutation frequency between forms may be due to dis- crepancies in the selection pressure they experience in their habitats [38]. Yet, given the typically greater pro- pensity for An. coluzzii to occur in agricultural habitats [39-42] later occurrence of 119S in An. coluzzii, either by independent mutation or introgression might be a more likely explanation. Unfortunately, as reported pre- viously for samples from other West African countries [43], sequence analysis of the resistant allele proved un- informative with respect to origin, owing to a complete lack of sequence variation. Unless there is very strong selection for the particular haplotype detected, this is more consistent with spread of a recent mutation than multiple origins or selection from standing variation. There is one intriguing additional possible explanation for the difference in 119S frequency between An. coluzzii and An. gambiae s.s. Although both insecticides appear to be effective in killing the Ace-1 homozygous wild-type individuals, a significant interaction was found between Ace-1 genotype and species, such that heterozygous indi- viduals are more resistant in An. gambiae s.s than An. coluzzii. The mechanistic reason for this is unclear but might involve the presence of additional, non-target site mechanisms or perhaps differences in the frequency or nature of Ace-1 duplication between molecular forms.

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“ADMET and Molecular Docking Studies of Novel 2, 4-dioxo-4-phenyl butanoic acid analogs as Mycobacterium Tuberculosis Inhibitors” by Manal Ali Elhag, Bakheit Mustafa, Obaid Bashier Ismail, Hanfi Mohd Zia, Nazar Mohammed Gabra, Salah Murghani, M.A. Baseer,

“ADMET and Molecular Docking Studies of Novel 2, 4-dioxo-4-phenyl butanoic acid analogs as Mycobacterium Tuberculosis Inhibitors” by Manal Ali Elhag, Bakheit Mustafa, Obaid Bashier Ismail, Hanfi Mohd Zia, Nazar Mohammed Gabra, Salah Murghani, M.A. Baseer, India.

Despite the availability of various techniques for diagnosis, the presence of improved and modified version of vaccines and the existence of more than a dozen of drugs, tuberculosis still remains as a significant infectious disease. The publication of complete genome of Mycobacterium tuberculosis has lead to the development of new genetic tools to ascertain the functioning of individual genes, leading to subsequent identification and validation of potential drug targets. With the help of Rational Drug designing, a computer-aided approach to find molecules with desired chemical and geometric properties that bind in a receptor cavity of specific target protein. A series of invention of 1HZP enzyme inhibitors lead to the discovery of 2, 4-dioxo-4-phenylbutanoic acid analogues. These analogues are more potent in binding with 1HZP enzyme. Aim of this study is to identify new compounds that can effectively inhibit 1HZP enzyme activity. Our methods include minimization of analogues with commercial software’s followed by docking using GOLD. From docking analysis, we found that substitutions like sulfonic acid and methoxy are more efficient in inhibiting the 1HZP activity. It is hoped that promising new drugs for the effective treatment of TB will be possible in the near future.

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Rapid multiplex gene expression assays for monitoring metabolic resistance in the major malaria vector Anopheles gambiae

Rapid multiplex gene expression assays for monitoring metabolic resistance in the major malaria vector Anopheles gambiae

The novel multiplex qRT-PCR assays for monitoring the expression levels of genes previously associated with metabolic resistance in An. gambiae (s.l.) are simple to perform, robust, rapid, cost-effective and work with RNAlater® preserved field-collected mosquitoes that can be shipped at room temperature. The approach we sug- gest here is, therefore, relevant and suitable for insecti- cide resistance management (IRM) programmes, on the one hand because large numbers of specimens can be handled without extra care, and on the other hand be- cause the speed of the assay allows for producing data as required within short time. The specificity of P450-based metabolic resistance against some but not other active ingredients, as well as the “ unexpected case ” of cross-resistance against different groups of insecticides [41], indicates that the development of diagnostic tools able to detect specific detoxification enzyme-based re- sistance is important. Together with bioassays and mo- lecular tests for target site resistance detection, our assays could identify resistance at an early stage and guide efficient and sustainable IRM strategies.

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Formulation Development and Evaluation of Transdermal Patch Containing Amlodipine Besylate

Formulation Development and Evaluation of Transdermal Patch Containing Amlodipine Besylate

transdermal permeation of a model β -blocker, propranolol, by fatty acid: a melting point depression effect and found that the binary mixture of propranolol and fatty acid, the addition compounds are formed by interaction between the carbonyl group of the β-blocker, to form a salt. The oppositely charged species of the salt have been shown to permeate the human epidermal membrane by an ion – pair mechanism. This is in agreement with the work by Green and Hadgraft (1987) which suggested the formation of ion-pairs between propranolol and fatty acids. Where the process was driven by a pH gradient.

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Mechanism of reversal of multidrug resistance by curcumin in human colorectal cancer cell line HCT-8/5-FU.

Mechanism of reversal of multidrug resistance by curcumin in human colorectal cancer cell line HCT-8/5-FU.

HCT-8/5-FU cells in log-phase growth were grown to 80% confluency and treated with different concentrations of curcumin and 5-FU. After culturing for 48 h at 37°C with 5% CO 2 , the culture medium was aspirated followed by washing of the cells thrice with phosphate-buffered saline. Cell lysis buffer was added to the pellets and mixed with a pipette to ensure complete cell disruption. Total protein from cells that received each treatment was extracted and 5X sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer was added to the extracted protein, followed by heat denaturation. After cooling, samples were centrifuged (8000 r/min) for 5 min, and the supernatants were collected. Proteins present in samples were separated by gel electrophoresis, which ran for 70 min, followed by transferring of the gel onto a membrane at 100 V. Membranes were blocked with 5% nonfat milk for 1 h, and then incubated overnight with primary antibody at 4°C. Incubation with secondary antibody was conducted for 1 h at 25°C, followed by thorough washing of the membranes. Developer solutions A and B were mixed, and 1 mL mixture was added onto the membrane. ChemiScope Mini chemiluminescence imaging system was used to detect and photograph the target protein bands. β-Actin was used as the control.

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Metallochaperones Are Needed for Mycobacterium tuberculosis and Escherichia coli Nicotinamidase-Pyrazinamidase Activity

Metallochaperones Are Needed for Mycobacterium tuberculosis and Escherichia coli Nicotinamidase-Pyrazinamidase Activity

relating to the organism’s survival in the host, have been identified in other pathogenic bacteria (66, 67). Regarding the possibility that Rv2059 (or other, similar metallochap- erones) is an alternative drug target that could have an effect similar to that of PZA, blocking Rv2059 with a specific drug would result in a less active PZAse and thus in PZA resistance. However, other, similar metallochaperones may be involved in the activation of essential metalloenzymes. In those cases, metallochaperones may constitute poten- tially important drug targets. Further studies are needed to confirm our findings and also to further our understanding of the importance of metallochaperones to mecha- nisms of mycobacterial resistance to PZA.

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Estimation of allele specific Ace 1 duplication in insecticide resistant Anopheles mosquitoes from West Africa

Estimation of allele specific Ace 1 duplication in insecticide resistant Anopheles mosquitoes from West Africa

droplet cluster, as visualized on each of the FAM and HEX probes. The ddPCR results were expressed as the number of droplets where amplification has or has not occurred (positive and negatives, respectively). In the case of this study, where a FAM/HEX duplex assay was performed, four discrete clusters of droplets are possible: (1) no target allele (negative FAM/negative HEX), indica- tive of a negative control or failed assay; (2, 3) only one of the targets is positive (negative FAM/positive HEX, or positive FAM/negative HEX), indicative of a homozy- gote; or (4) both targets are positive (positive FAM/posi- tive HEX), indicative of a heterozygote. Results from (2), (3) and (4) can be used to compute the average number of copies of PCR amplicons for each allele (λ R

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Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda

Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda

To investigate if metabolic detoxification also plays a role in the resistance of the OP and PYR strains we designed a microarray based on all available S. frugiperda EST sequences and used it to compare gene expression in the resistant strains with a susceptible reference strain. A number of ESTs that can be considered potential candidates for a role in insecticide resistance, were shown to be overexpressed in the resistant strains. These included ESTs encoding GSTs, P450s and CEs, enzymes that have been implicated in metabolic resistance in many arthropod species. Several other ESTs that encode enzymes capable of metabolizing xenobiotics (and potentially insecticides) including, short chain dehydrogenases, aldehyde dehydroxygenases and glucosyl-glucur- onosyl transferases were also overexpressed. In the case of the OP strain of particular relevance was a sequence encoding a CE that was identified as being significantly over-expressed in both microarray (21-fold) and qPCR (,11-fold) experiments. Overex- pression of E4 esterase was first described in M. persicae where it confers broad-spectrum resistance to organophosphates and carbamates as a consequence of both sequestration and ester hydrolysis [38]. Purification of this esterase and further functional analysis is therefore warranted. Several ESTs encoding P450s were also overexpressed in the OP strain in microarray analyses and one of these (Sf2M09131-5-1) was confirmed as overexpressed by qPCR ,3-fold. This EST encodes a P450 with highest sequence similarity to Spodoptera littoralis CYP6B50. Members of the P450 CYP6 family have been shown to confer resistance to organo- phosphates in several insects previously [13]. Further suggestion that this P450 may be involved in resistance is that the EST was derived from a xenobiotic induced midgut S. frugiperda library. Several ESTs encoding GSTs were significantly overexpressed in the OP strain in microarray analysis. Q-PCR revealed that two of these were overexpressed (,3-5-fold) and belong to the epsilon

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Insecticide resistance mediated 1 by an exon skipping event

Insecticide resistance mediated 1 by an exon skipping event

Beyond splice factors, transcriptome profiling of the Spin and SpinSel strains revealed very few genes over- expressed in the SpinSel strain belonging to the major superfamilies of enzymes frequently implicated in metabolic resistance in insects. For example, no tran- scripts encoding cytochrome P450s (P450s), car- boxylesterases (CEs) or UDP-glucuronosyltransferase (UGTs) were identified as overexpressed in reads mapped to either assembly 1 or 2 (Table S4/S5, Sup- porting information). A single transcript encoding a glutathione S-transferase (GST) (comp155108_c1_seq10) was identified as upregulated (17-fold by DESeq2) in the SpinSel strain in reads mapped to assembly 2 (Table S5, Supporting information). However, this tran- script encodes a conserved GST enzyme belonging to the zeta class that corresponds to GST zeta 1 (GSTZ1), an enzyme catalysing maleylacetoacetate to fumarylace- toacetate in the penultimate step of the phenylalanine/ tyrosine degradation pathway (Fern andez-Ca~ n on & Pe~ nalva 1998), and is not known to play a role in the metabolism of xenobiotics. A single transcript (com- p62327_c1_seq6) encoding an aldo-keto reductase, an enzyme family known to play an important role in the phase II detoxification of several drugs and xenobiotics in humans, was identified as overexpressed (7.4-fold) in the assembly 1 comparison (Table S4, Supporting infor- mation); however, an almost identical transcript (com- p145080_c0_seq1) of the same gene (99% sequence identity) was identified as downregulated in the assem- bly 2 comparison (Table S5, Supporting information). Finally, no transcripts with potential roles in phase III metabolism (i.e. ABC transporters) were overexpressed in the SpinSel strain in either comparison. In summary, RNAseq analysis provided little evidence of additional, non-target-site, mechanisms of resistance in the SpinSel strain.

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Isolation of Rifampin Resistant Mutants ofListeria monocytogenes and Their Characterization byrpoB Gene Sequencing, Temperature Sensitivity for Growth, and Interaction with an Epithelial Cell Line

Isolation of Rifampin Resistant Mutants ofListeria monocytogenes and Their Characterization byrpoB Gene Sequencing, Temperature Sensitivity for Growth, and Interaction with an Epithelial Cell Line

mutations, they showed slightly different properties of binding to Caco-2 cells; this indicates that a second mutation may have occurred in one of the mutants during the one-step selection procedure, which may be present either in a part of the rpoB gene not sequenced in this study or elsewhere on the genome. The ability of rifampin to penetrate host cells could become particularly important for the treatment of listeriosis, since L. monocytogenes is known to remain intracellular and spread from cell to cell by an actin-based motility process (12). Be- cause antibiotic resistance in agriculture is a growing problem, it will be increasingly important to identify mutant organisms, their genotypes, and their altered phenotypes in order to un- derstand their dissemination.

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An Evolutionarily Conserved Mechanism for Intrinsic and Transferable Polymyxin Resistance

An Evolutionarily Conserved Mechanism for Intrinsic and Transferable Polymyxin Resistance

labeled substrate NDB-glycerol-3-PEA (1-acyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl}-sn-glycero-3-phosphoethanolamine) (Fig. 4I and 5A to C) (45). Thin-layer chromatography (TLC) assays suggested that (i) 3 mutants of MCR-2 (N106A, T110A, and S328A) retain partial enzymatic activities similar to that of the wild-type MCR-2 in that an intermediate product, NBD-glycerol, is produced (Fig. 4I), while (ii) the other 9 MCR-2 point mutants have no detectable activity, as evidenced by NBD-glycerol being absent and only the substrate, NBD-glycerol-3-PEA, being observed, almost identical to the results for the negative controls (Fig. 4I). The differences in the enzymatic activities of MCR-2 mutants were further demonstrated through their ability to modify lipid A anchored on the bacterial outer membrane, as determined using MALDI-TOF MS (Table 1). The presence of wild-type MCR-2 gives a new peak (m/z, 1,919.991), corresponding to the production of PPEA-1(4=)-lipid A, when compared to the profile obtained for E. coli MG1655, which has only one dominant lipid A peak (m/z, 1,796.243) (Table 1). This validates its role in catalyzing the enzymatic transfer of PE from PE lipids to lipid A in vivo. Consistent with observations made during the TLC trials, only 3 of the 12 MCR-2 mutants retained partial PEA transfer activity, giving a specific peak for PPEA-1(4=)-lipid A (m/z, 1,919.114 to 1,919.857) (Table 1). The other 9 mutants of MCR-2 lost enzymatic activity because of functional impairment of the cavity occupied by the PE lipid molecule (Table 1). This highlights the importance of cavity- forming residues in the biochemistry of MCR-2, a transferable determinant of colistin resistance, closely matching EptA, an intrinsic determinant of colistin resistance.

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Antimalarial drug resistance

Antimalarial drug resistance

Malaria is a hematoprotozoan parasitic infection transmitted by cer- tain species of anopheline mosquitoes (Figure 1). Four species of plasmodium commonly infect humans, but one, Plasmodium falci- parum, accounts for the majority of instances of morbidity and mor- tality. There has been a resurgence of interest in malaria in recent years as the immensity of the burden it imposes on poor countries in the tropics has become apparent, and as efforts at control have foundered after the failure of the global eradication campaign in the 1960s. Control has traditionally relied on two arms: control of the anopheline mosquito vector through removal of breeding sites, use of insecticides, and prevention of contact with humans (via the use of screens and bed nets, particularly ones that are impregnated with insecticides); and effective case management. A long-hoped-for third arm, an effective malaria vaccine, has not materialized and is not expected for another decade. Case management has relied largely on antimalarials (mainly chloroquine, and more recently sulfadoxine- pyrimethamine [SP]), which are inexpensive and widely available and are eliminated slowly from the body. Together with antipyret- ics, antimalarials are among the most commonly used medications in tropical areas of the world. Misuse is widespread. In many parts of the tropics, the majority of the population has detectable con- centrations of chloroquine in the blood. The extensive deployment of these antimalarial drugs, in the past fifty years, has provided a tremendous selection pressure on human malaria parasites to evolve mechanisms of resistance (Table 1). The emergence of resistance, particularly in P. falciparum, has been a major contributor to the global resurgence of malaria in the last three decades (1). Resistance is the most likely explanation for a doubling of malaria-attributable child mortality in eastern and southern Africa (2).

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INDUCIBLE CLINDAMYCIN RESISTANCE IN STAPHYLOCOCCUS AUREUS: A STUDY IN TERTIARY CARE HOSPITAL IN BHOPAL

INDUCIBLE CLINDAMYCIN RESISTANCE IN STAPHYLOCOCCUS AUREUS: A STUDY IN TERTIARY CARE HOSPITAL IN BHOPAL

Introduction: Emergence of increasing resistance in Staphylococcus aureus has renewed the interest in Clindamycin (lincosamide) usage because of its good pharmacokinetic properties in the treatment of Staphylococcal infections especially in Methicillin Resistant Staphylococcus aureus (MRSA) infections. Erythromycin (macrolide) also used to treat Staphylococcal infections but it is a potent inducer of Clindmycin resistance. Combined use of these drugs pose a threat of treatment failure. Phenotypic detection of Erythromycin induced Clindamycin resistance by a simple D- test can be done in clinical laboratories to curb inappropriate use of these drugs in Staphylococcal infections including MRSA strains. Objectives: To find out the burden of Inducible clindamycin resistance & its relation with MRSA. Materials and Methods: A total of 127 clinical isolates of S. aureus between March 2013- July2013 collected from various Clinical samples were processed to routine antimicrobial Suceptibility test using modified Kirby- Bauer disc diffusion method-using Cefoxitin (30 μg) disc to detect MRSA. Erythromycin (15 μg) & Clindamycin (2 μg) disc placed at a distance of 15mm to detect Erythromycin induced Clindamycin resistance by D – test as per CLSI guidelines. Results: Among 127 isolates, 21(16.53%) isolates showed inducible Clindamycin resistance, out of which 13 were MRSA strains. So percentage of inducible Clindamycin resistance was higher in MRSA (61.90%) as compared to MSSA (38.09%). Conclusions: For optimum treatment of patients, D-test which is simple and feasible test should be used as routine lab method to detect inducible Clindamycin resistance in Staphylococci.

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Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

Data from catalytic assays, modelling and X-ray crystallography suggested that one of the non-synonymous changes had a marked effect on DDTase activity. A TaqMan SNP genotyping assay was designed to screen for the mutation in individual mosquitoes (see Table S1 in File S1 for primer and probe sequences). DNA extracts from adult female mosquitoes from a number of locations in sub-Saharan Africa were genotyped for the Gste2 allelic variants. In addition female mosquitoes with known DDT susceptibility phenotypes, as defined by the standard WHO protocol, were obtained from Burkina Faso[49] and Benin. SNP genotyping assays were performed in 10 ml volume containing 1x Sensimix (Bioline), 1x primer/probe mix and 1 ml template DNA with a temperature profile of 95uC for 10min followed by 40 cycles of 92uC for 15s and 60uC for 1min on an Agilent MX3005 real-time PCR machine. VIC and FAM fluorescence was captured at the end of each cycle and genotypes called from endpoint fluorescence using the Agilent MXPro software. Specimens from Benin and Burkina Faso were also screened for known DDT-resistance associated variants in the voltage-gated sodium channel [5,50]. Genotype: phenotype associations were assessed using a general- ized linear model with a logit link function [42], chi-squared tests Poptools 3.2 [51], and sample haplotype frequencies estimated using Haploview 4.2 [52].

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