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Abstract: The sphingosine-1-phosphate type 1 (S1P1) receptor is a new target in the treatment of auto-immune diseases as evidenced by the recent approval of FTY720 (Fingolimod). The ligand-binding pocket of the S1P1 receptor has been generally characterised but detailed insight into ligand-specific differences is still lacking. The aim of the current study is to determine differences in ligand-induced S1P1 receptor activation using an in silico guided site-directed mutagenesis strategy. S1P1 mutant receptors (modifications of residues Y982.57, R1203.28, F1253.33) were probed with a chemically diverse set of S1P1 agonists (S1P, dihydro-S1P (dhS1P), R-, S- and racemic FTY720-P, VPC24191, SEW2871). Mutation of the R3.28 residue generally results in a reduction of the potency of all ligands although the synthetic ligands including FTY720-P are less sensitive to these mutations. The Y2.57F mutation does not affect the potency of any of the ligands tested, but for all ligands except FTY720-P a significant decrease in potency is observed at the Y2.57A mutant. The F3.33A mutation significantly decreased the potency of FTY720-P and is detrimental for SEW2871 and VPC24191. The non-aromatic endogenous ligands S1P and dhS1P are less affected by this mutation. Our in silico guided mutagenesis studies identified new molecular determinants of ligand-induced S1P1 receptor activation: 1) the flexibility of the polar head of the agonist to maintain a tight H-bond network with R3.28 and 2) the ability of the agonist to make aromatic π-stacking interactions with F3.33. Interestingly, FTY720-P has both chemical properties and is the only ligand that can efficiently activate the Y2.57A mutant. [Copyright &y& Elsevier]

Objectives To describe a novel extended-spectrum oxacillinase, named OXA-145, differing from narrow-spectrum OXA-35 (from the OXA-10 group) by deletion of residue Leu-165. The genetic environment of blaOXA-145 and the biochemical properties of OXA-145 are reported. We also assessed the impact of the Leu-165 deletion on the hydrolysis spectrum of the ancestor OXA-10. Methods Extended-spectrum β-lactamase OXA-145 was identified in the multidrug-resistant clinical Pseudomonas aeruginosa 08-056, and characterized by isoelectric focusing, PCR and DNA sequencing. Antibiotic susceptibility tests were performed by agar dilution. The resistance profiles conferred by cloned blaOXA-10, blaOXA-35, blaOXA-145 and a blaOXA-10 derivative obtained by site-directed mutagenesis were determined in Escherichia coli. Kinetic parameters of OXA-35 and OXA-145 were established after purification of His-tagged proteins. Results The sequence of OXA-145, encoded by a class 1 integron-borne gene in strain 08-056, differed from that of narrow-spectrum penicillinase OXA-35 by a single amino acid deletion (Leu-165) located in the highly conserved omega loop. Deletion of Leu-165 from OXA-35 (yielding OXA-145) or OXA-10 (the progenitor of OXA-35) extended the hydrolysis spectrum to third-generation cephalosporins and to monobactams, while reducing that for penicillins. OXA-145 showed biphasic hydrolysis curves for all the substrates tested. Its activity against nitrocefin was 10-fold higher in the presence of sodium hydrogen carbonate. Conclusions OXA-145 is a new extended-spectrum β-lactamase from the OXA-10 group. The deletion of Leu-165 is responsible for a shift in the hydrolysis spectrum from penicillins to third-generation cephalosporins, as well as monobactams. The loss of penicillin hydrolysis was due to a non-carboxylated Lys-73. [ABSTRACT FROM PUBLISHER]

Dialelic crosses and backcrosses of pyrethroid resistant (RR) and susceptible (SS) Rhipicephalus ( Boophilus) microplus tick strains were carried out and the substitution (Phe-Ile) within the sodium channel gene was monitored in order to analyze the effects of the genotype on the pyrethroid resistance phenotype as measured by the larval packet test (LPT). Parental strains: susceptible (SS) and resistant (RR); dialelic crosses: RS (♂RR × ♀SS), and SR (♂SS × ♀RR); and backcrosses: RS × SS, RS × RR, SR × SS and SR × RR were infested on 280 kg calves. Resistance type (monogenic or polygenic) and effective dominance were determined based on the discriminant concentration (DC) for cipermethrine (0.5%), deltamethrine (0.09%) and flumethrine (0.01%). Allele specific PCR (AS-PCR) was used for genotyping, looking at a sodium channel mutation (Phe-Ile substitution). The mortality rates and allele frequency of susceptible and pyrethroid resistant reference strains were 0% mortality and 90% RR alleles for resistant strain, and 100% mortality and 0% RR alleles as measured by the larval packet test (LPT) and allele specific PCR (AS-PCR) respectively. Backcrossed strain SR × RR showed an effective dominance (D) of 0.605 for cypermethrin, 0.639 for deltamethrin and 0.498 for flumethrin, while survival of backcrosses RS × SS, RS × RR and SR × SS showed a significant tendency to recesivity. Backcrossed strain SR × RR (69.4%) also showed a higher RR genotype frequency with regards to RS × SS (25.5%), RS × RR (36.7%) and SR × SS (32.0%), however, susceptible allele was inherited in general as an incomplete dominant trait. Monogenic inheritance hypothesis was tested and the results showed monogenic inheritance for cypermethrin and flumethrin ( P < 0.05) but not for deltamethrin ( P > 0.05). However, significant correlation was found between RR genotype and the survival rate for all three pyrethroids used ( P < 0.05), suggesting that a single substitution on the sodium channel gene can be responsible for resistance to pyrethroids as a class, due to the high frequency for RR genotypes. Combination with different mutations or metabolic resistance mechanisms cannot be excluded. [ABSTRACT FROM AUTHOR]

Background: Clarithromycin resistance in Helicbacter pylori has been found to be associated with point mutations in 23s rRNA gene leads to reduced affinity of the antibiotic to its ribosomal target or changing the site of methylation. The aim of this study was to determine the most important point mutations in 23s rRNA gene in H. pylori that are closely related to clarithromycin resistance among such isolates. Methods: Sixty three H. pylori isolates, obtained from gastric biopsy speciemens in Kerman, Iran, were used to evaluate their susceptibility to clarithromycin by disk diffusion test, and to detect the most common point mutations in 23s rRNA gene associated with clarithromycin resistance by Polymerase chain reaction-amplification and restriction fragment length polymorphism (PCR-RFLP) and 3'-mismatch PCR. Results: 31.7% of the H. pylori isolates were resistant to clarithromycin, and each of the resistant isolate had at least one of the most common point mutations in 23s rRNA gene associated with calrithromycin resistance. Conclusion: According to our results three common point mutation in 23s rRNA gene in H. pylori are closely related to clarithromycin resistance. There was an absolute relation between 23s rRNA gene point mutations and clarithromycin resistance in this study. Helicbacter pylori resistance to clarithromycin can cause failure in the eradications of the bacteria. The resistance of the bacteria is expanding in most parts of the world including Iran. [ABSTRACT FROM AUTHOR]

Genetic lesions are crucial for cancer initiation. Recently, whole genome sequencing, using next generation technology, was used as a systematic approach to identify mutations in genomes of various types of tumors including melanoma, lung and breast cancer, as well as acute myeloid leukemia (AML). Here, we identify tumor-specific somatic mutations by sequencing transcriptionally active genes. Mutations were detected by comparing the transcriptome sequence of an AML sample with the corresponding remission sample. Using this approach, we found five non-synonymous mutations specific to the tumor sample. They include a nonsense mutation affecting the RUNX1 gene, which is a known mutational target in AML, and a missense mutation in the putative tumor suppressor gene TLE4, which encodes a RUNX1 interacting protein. Another missense mutation was identified in SHKBP1, which acts downstream of FLT3, a receptor tyrosine kinase mutated in about 30% of AML cases. The frequency of mutations in TLE4 and SHKBP1 in 95 cytogenetically normal AML patients was 2%. Our study demonstrates that whole transcriptome sequencing leads to the rapid detection of recurring point mutations in the coding regions of genes relevant to malignant transformation. [ABSTRACT FROM AUTHOR]

Background: Helicobacter pylori is a fastidious microorganism and therefore standard phenotypic susceptibility tests can take at least 10-14 days. Molecular based diagnostic assays offer an alternative approach to obtain susceptibilities to antibiotics and detection of point mutations with greater accuracy. The aim of this study was the assessment of Clarithromycin resistance and its point mutations by using real-time PCR assay. Methods: This cross-sectional descriptive study was performed on 200 gastric biopsy specimens obtained from patients undergoing upper gastrointestinal endoscopy at Hajar hospital in Shahrekord. Initially Helicobacter pylori were identified by rapid urease test (RUT) and polymerase chain reaction (PCR). Then clarithromycin resistance and its point mutations were evaluated by using specific probes and real-time PCR technique. Findings: Of total samples, 164 (82%) were Helicobacter pylori positive. Overall, a clarithromycin susceptible strains were detected in 105 (64.02%) patients and resistance strains were detected in 59 (35.98%) which were identified as 4 (2.44%) A 2144G, 26 (15.85%) A2143G, 15 (9.15%) A2143C, and 20 (12.19%) A2142G point mutations. Genotype of 5 (8.47%) strains was not detected. Purely resistant strains were detected in 38 (23.17%), while heteroresistant were found in the remaining 16 cases (9.76%). Conclusion: Results showed that real-time PCR assay has high accuracy to simultaneously identify Helicobacter pylori and clarithromycin resistance types directly in gastric biopsy specimens in short time. [ABSTRACT FROM AUTHOR]

We describe a new nondeletional αα-thalassemia (αα-thal) determinant found in a Moroccan infant and in two members of his family. The new mutation generates an abnormal hemoglobin (Hb) as a consequence of a Pro→→Ser amino acid substitution at codon 37 (old nomenclature) of the αα2 gene. The new Hb variant is barely separable on high performance liquid chromatography (HPLC) but the expression of the αα chain mutant measured on reversed phase chromatography is one-third of that expected from a stable αα2 variant, which explains the mild αα-thal phenotype observed in the carriers. As shown for other mutations described in our laboratory (i.e., Hb Gouda), this variant could also be common in the North African population, overlooked because of the mild phenotype and silent behavior on HPLC. Nevertheless, these silent variants could generate intermediate Hb H diseases in association with Mediterranean αα0-thal deletion defect. [ABSTRACT FROM AUTHOR]

Triple therapy is commonly used for the treatment of Helicobacter pylori infection. We determined risk factors associated with its failure in compliant patients focusing on H. pylori density, virulence marker and 23S ribosomal RNA (rRNA) point mutations associated with clarithromycin resistance. H. pylori infection was diagnosed by 14C urea breath test (14C UBT) and rapid urease test or histology. Triple therapy with esomeprazole 20 mg b.i.d., amoxicillin 1 g b.i.d. and clarithromycin 500 mg b.i.d. was prescribed for 10 days. 14C UBT was repeated 4 weeks after treatment. In total, 111 patients [69 (62%) males] with a mean age of 46±16 years were enrolled. The mean age of treatment failure was 39±14 years compared to 48±16 years with eradication (P=0·002). Treatment failure was associated with younger mean age, point mutations in the 23S rRNA gene of H. pylori and vacA s1a and m1 when associated with cagA negativity. [ABSTRACT FROM AUTHOR]

Abstract: Phalaris paradoxa is a competitive grass commonly found in durum wheat crops of central and southern Italy. Among the 85 populations screened from 1998 to 2008 for resistance to ACCase-inhibitors and graminicide sulfonylureas, 17 resulted as being resistant to at least one ACCase inhibitor while none of the populations showed resistance to sulfonylureas. ACCase resistance in hood canary-grass seems to be spreading rather slowly in Italy. Out of the 17 populations, seven were characterised through outdoor dose-response pot experiments to investigate resistance levels and cross-resistance patterns to ACCase-inhibitors and multiple resistance to other mode of action. Molecular bases of resistance to the recently introduced DEN herbicide pinoxaden were also investigated. Six populations were confirmed to be ACCase-resistant with various cross-resistance patterns. Two populations were resistant to all tested ACCase herbicides, with pinoxaden resistance indexes (RI) based on survival ranging from 22 to 50. The two populations have been molecularly characterised for resistance to pinoxaden. A single point-mutation in the ACCase gene was identified in each population, causing the amino-acid substitutions of Ile1781Val and Asp2078Gly in 0478L and 0025, respectively. The results suggest that resistance of P. paradoxa to pinoxaden is due to an altered target site and different mutations cause different resistance levels. The biological characteristics of the species, mainly self-pollinated, and the absence of multiple resistance allow herbicides with different modes of action to be used for controlling ACCase-resistant populations. Chemical tools should be carefully used within integrated weed management strategies. [Copyright &y& Elsevier]

Abstract: In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency. [Copyright &y& Elsevier]

Abstract: We consider mixtures of two DNA sequences t and t′ differing by a single nucleotide, which are analyzed by an Agilent custom DNA microarray. In particular we focus on the case in which t, the “wild type”, is predominantly abundant and t′ the “mutant” is at very low concentrations compared to t. We show that by using appropriately designed arrays it is possible to accurately quantify the presence of t′ even at low relative concentrations (≈1%). The detection method is based on thermodynamic models of DNA hybridisation and on the analysis of a large number of hybridisation intensities from probes containing one or two mismatches with respect to t and t′. [ABSTRACT FROM AUTHOR]

Abstract: A rational design approach was used to create the mutant Candida antarctica lipase B (CALB, also known as Pseudozyma antarctica lipase B) V190A having a k cat three times higher compared to that of the wild type (wt) enzyme for the transacylation of the industrially important compound methyl methacrylate. The enzymatic contribution to the transacylation of various acrylates and corresponding saturated esters was evaluated by comparing the reaction catalysed by CALB wt with the acid (H2SO4) catalysed reaction. The performances of CALB wt and mutants were compared to two other hydrolases, Humicola insolens cutinase and Rhizomucor mihei lipase. The low reaction rates of enzyme catalysed transacylation of acrylates were found to be caused mainly by electronic effects due to the double bond present in this class of molecules. The reduction in rate of enzyme catalysed transacylation of acrylates compared to that of the saturated ester methyl propionate was however less than what could be predicted from the energetic cost of breaking the π-system of acrylates solely. The nature and concentration of the acyl acceptor was found to have a profound effect on the reaction rate. [Copyright &y& Elsevier]

Abstract: The two-spotted spider mite Tetranychus urticae Koch is one of the economically most important pests in a wide range of outdoor and protected crops worldwide. Its control has been and still is largely based on the use of insecticides and acaricides. However, due to its short life cycle, abundant progeny and arrhenotokous reproduction, it is able to develop resistance to these compounds very rapidly. As a consequence, it has the dubious reputation to be the“most resistant species” in terms of the total number of pesticides to which populations have become resistant, and its control has become problematic in many areas worldwide. Insecticide and acaricide resistance has also been reported in the ectoparasite Sarcoptes scabiei, the causative organism of scabies, and other economically important Acari, such as the Southern cattle tick Rhipicephalus microplus, one of the biggest arthropod threats to livestock, and the parasitic mite Varroa destructor, a major economic burden for beekeepers worldwide. Although resistance research in Acari has not kept pace with that in insects, a number of studies on the molecular mechanisms responsible for the resistant phenotype has been conducted recently. In this review, state-of-the-art information on T. urticae resistance, supplemented with data on other important Acari has been brought together. Considerable attention is given to the underlying resistance mechanisms that have been elucidated at the molecular level. The incidence of bifenazate resistance in T. urticae is expanded as an insecticide resistance evolutionary paradigm in arthropods. [Copyright &y& Elsevier]

Leukemias are a heterogeneous group of malignant blood diseases that are characterized by expansion of immature blast cells. The point molecular mechanisms of leukemogenesis are still unknown. Leukemia patients frequently have mutations of the genes responsible for normal proliferation and differentiation of hematopoietic stem cells. At present, scientific groups worldwide are engaged in biomedical studies of the structural and functional aspects of leukemic oncogenes and their role in human and animal leukemogenesis. The review describes the current concepts of the molecular properties of oncogenes whose activation may lead to CBF-AML, which results from mutations of the genes for the core binding factors AML1 (CBF6h) and CBFß. [ABSTRACT FROM AUTHOR]

Objectives: An in vivo chloroquine efficacy study was undertaken on the island of Car Nicobar because a temporal rise in the Plasmodium falciparum parasite population containing mutations in the chloroquine resistance transporter (PfCRT) protein has been reported there. [ABSTRACT FROM PUBLISHER]

Abstract: Point mutations emerge as one of the rate-limiting steps in tumor response to small molecule inhibitors of protein kinases. Here we characterized the response of the MET mutated variants, V1110I, V1238I, V1206L and H1112L to the small molecule SU11274. Our results reveal a distinct inhibition pattern of the four mutations with IC50 values for autophosphorylation inhibition ranging between 0.15 and 1.5μM. Differences were further seen on the ability of SU11274 to inhibit phosphorylation of downstream MET transducers such as AKT, ERK, PLCγ and STAT3 and a variety of MET-dependent biological endpoints. In all the assays, H1112L was the most sensitive to SU11274, while V1206L was less affected under the used concentration range. The differences in responses to SU11274 are discussed based on a structural model of the MET kinase domain. [Copyright &y& Elsevier]

Abstract: The important and diverse biological functions of adrenergic receptors, a subclass of G protein-coupled receptors (GPCRs), have made the search for compounds that selectively stimulate or inhibit the activity of different adrenergic receptor subtypes an important area of medicinal chemistry. We previously synthesized 2-, 5-, and 6-fluoronorepinehprine (FNE) and 2-, 5-, and 6-fluoroepinephrine (FEPI) and found that 2FNE and 2FEPI were selective β-adrenergic agonists and that 6FNE and 6FEPI were selective α-adrenergic agonists, while 5FNE and 5FEPI were unselective. Agonist potencies correlated well with receptor binding affinities. Here, through a combination of molecular modeling and site-directed mutagenesis, we have identified N293 in the β2-adrenergic receptor as a crucial residue for the selectivity of the receptor for catecholamines fluorinated at different positions. [Copyright &y& Elsevier]

Abstract: A hybrid network of evolutionary processors (an HNEP) is a graph where each node is associated with an evolutionary processor (a special rewriting system), a set of words, an input filter and an output filter. Every evolutionary processor is given with a finite set of one type of point mutations (an insertion, a deletion or a substitution of a symbol) which can be applied to certain positions of a string over the domain of the set of these rewriting rules. The HNEP functions by rewriting the words that can be found at the nodes and then re-distributing the resulting strings according to a communication protocol based on a filtering mechanism. The filters are defined by certain variants of random-context conditions. HNEPs can be considered as both language generating devices (GHNEPs) and language accepting devices (AHNEPs). In this paper, by improving the previous results, we prove that any recursively enumerable language can be determined by a GHNEP and an AHNEP with 7 nodes. We also show that the families of GHNEPs and AHNEPs with 2 nodes are not computationally complete. [Copyright &y& Elsevier]

Abstract: Clonal emergence of group A streptococci (GAS) with reduced susceptibility to fluoroquinolones (FQs) has been increasingly reported. Non-susceptibility is associated with various point mutations in the target-encoding genes and has only been described in a few emm types. We used a well-characterised GAS clinical paediatric collection from Brussels (Belgium) and Brasília (Brazil) to analyse the molecular basis of FQ non-susceptibility. GAS strains were tested for ciprofloxacin susceptibility and were screened for mutations in DNA gyrase- and topoisomerase IV-encoding genes. Genetic relationships between the different emm types were assessed by phylogenetic analysis of the whole surface-exposed part of the M protein. A high proportion (22.5%) of ciprofloxacin-non-susceptible isolates (minimal inhibitory concentration ≥2mg/L) was found among the Belgian strains. They belonged mostly to emm type 6 (87%). In Brazil, 6% of the isolates, belonging to seven distantly related emm types, were non-susceptible. Our phylogenetic analysis showed that non-susceptibility may arise in various genetic backgrounds. Sequence comparison of the quinolone resistance-determining regions (QRDRs) of the ParC- and ParE-encoding genes from susceptible and non-susceptible isolates revealed that most of the mutations were found in both classes of isolates, indicating an emm type-linked polymorphism. In conclusion, we observed a clonal spreading of non-susceptible emm type 6 GAS strains in Brussels and a polyclonal distribution of non-susceptible isolates in Brazil. All the Brazilian and Belgian emm type 6 strains displayed a S79A/F mutation in parC that convincingly explains the non-susceptible phenotype. [Copyright &y& Elsevier]

Abstract: Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1× Tris–borate–EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250 000) under reverse polarity with 15°C and 30°C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations. [Copyright &y& Elsevier]

The essence of reproduction involves propagation of genetic information from parents to offspring. In mammals, the frequency of spontaneously acquired mutations is lower in germ-line cells than in somatic cells, reflecting the role played by germ-line cells in the propagation of genetic information and the importance of maintaining genetic integrity in these cells. The Big Blue® transgenic mouse model was used to investigate the frequency and spectrum of: (i) spontaneous point mutations in germ cells as they develop naturally during the life cycle of the mouse; and (ii) acquired mutations that are normally transmitted from parents to offspring during natural and assisted reproduction. The study found that germ cells normally maintain a frequency of spontaneous point mutations that is 5-10-fold lower than that observed in somatic cells from the same individual, leading to embryos with very low frequencies of point mutations in the next generation. No significant differences in the frequency or spectrum of mutations between naturally conceived fetuses and assisted-conception fetuses were observed, indicating that, with respect to maintenance of genetic integrity, these methods are safe. Preliminary analysis of fetuses produced by somatic cell nuclear transfer indicates that maintenance of genetic integrity is regulated in a tissue-specific manner by epigenetic mechanisms that are subject to reprogramming during cloning. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms defined by morphologic dysplasia, peripheral cytopenia and clonal instability with enhanced risk of transformation into acute myeloid leukemia. The prognosis and clinical picture in MDS vary depending on patient-related factors (age, gender, comorbidity), the disease variant, cell types affected and genes involved in the malignant process. In fact, more and more data suggest that cytogenetic and molecular defects and gene variants are associated with the clinical course and prognosis in MDS. Although certain molecular defects are indicative of distinct cytogenetic abnormalities, others represent point mutations in critical target genes (RUNX1, N-RAS, JAK2, KIT, others) and sometimes are associated with a particular type of MDS, an overlap disease, a co-existing hematopoietic neoplasm or disease progression. Although most are somatic mutations, germ line mutations and gene polymorphisms have also been described in MDS. Some of these mutations may influence the natural course of disease, iron accumulation or disease progression. The present article provides a summary of our current knowledge about molecular and genetic markers in MDS, with special reference to their potential prognostic and therapeutic implications. [ABSTRACT FROM AUTHOR]

Coronaviruses (CoVs) constitute a large and diverse subfamily of positive-sense single-stranded RNA viruses. They are found in many mammals and birds and have great importance for the health of humans and farm animals. The current SARS-CoV-2 pandemic, as well as many previous epidemics in humans that were of zoonotic origin, highlights the importance of studying the evolution of the entire CoV subfamily in order to understand how novel strains emerge and which molecular processes affect their adaptation, transmissibility, host/tissue tropism, and patho non-homologous genicity. In this review, we focus on studies over the last two years that reveal the impact of point mutations, insertions/deletions, and intratypic/intertypic homologous and non-homologous recombination events on the evolution of CoVs. We discuss whether the next generations of CoV vaccines should be directed against other CoV proteins in addition to or instead of spike. Based on the observed patterns of molecular evolution for the entire subfamily, we discuss five scenarios for the future evolutionary path of SARS-CoV-2 and the COVID-19 pandemic. Finally, within this evolutionary context, we discuss the recently emerged Omicron (B.1.1.529) VoC. [ABSTRACT FROM AUTHOR]

Human Cytochrome P450 3A4 is a major P450 enzyme in the liver and gastrointestinal tract. It plays an important role in the metabolism of a wide variety of drugs, some endogenous steroids, and harmful environmental contaminants. To investigate the interindividual variation in CYP3A4 levels we have carried out a study on the genetic polymorphism of 100 breast cancer subjects using fine needle aspiration cytology (FNAC) sampling procedure. DNA was extracted from all the samples and PCR was carried out for detecting the CYP3A4 gene polymorphisms. We selected exon-7 and 10 which are present on the 5'-flanking coding region of the gene using the respective primers for PCR followed by direct automated sequencing method for detecting the mutations. These mutations were compared to the wild type sequence structures obtained from GenBank database (accession no. AF209389). We found two novel point mutations which are heterozygous mutant alleles. The two variant alleles were Ile222Arg and Phe175Val occurring in coding region of exon-7. Ile222Arg mutation was found in 3 malignant cases whereas Phe175Val mutation was found in 4 malignant cases. This is the first report of these two novel point mutations in CYP3A4 gene. These mutations in the gene in respective patients were found to relate to drug response in invasive ductal carcinomas of breast cancer. [ABSTRACT FROM AUTHOR]

It is believed that the codon–amino acid assignments of the standard genetic code (SGC) help to minimize the negative effects caused by point mutations. All possible point mutations of the genetic code can be represented as a weighted graph with weights that correspond to the probabilities of these mutations. The robustness of a code against point mutations can be described then by means of the so-called conductance measure. This paper quantifies the wobble effect, which was investigated previously by applying the weighted graph approach, and seeks optimal weights using an evolutionary optimization algorithm to maximize the code's robustness. One result of our study is that the robustness of the genetic code is least influenced by mutations in the third position—like with the wobble effect. Moreover, the results clearly demonstrate that point mutations in the first, and even more importantly, in the second base of a codon have a very large influence on the robustness of the genetic code. These results were compared to single nucleotide variants (SNV) in coding sequences which support our findings. Additionally, it was analyzed which structure of a genetic code evolves from random code tables when the robustness is maximized. Our calculations show that the resulting code tables are very close to the standard genetic code. In conclusion, the results illustrate that the robustness against point mutations seems to be an important factor in the evolution of the standard genetic code. [ABSTRACT FROM AUTHOR]

• Two ethylmethanesulfonate (EMS) mutant populations of the semi-winter rapeseed cv. Ningyou7 were constructed with high mutant load, to provide a TILLING platform for functional genomics in Brassica napus, and for introduction of novel allelic variation in rapeseed breeding. • Forward genetic screening of mutants from the M2 populations resulted in identification of a large number of novel phenotypes. Reverse genetic screening focused on the potentially multi-paralogous gene FAE1 (fatty acid elongase1), which controls seed erucic acid synthesis in rapeseed. A B. napus BAC library was screened, and loci in a reference mapping population (TNDH) were mapped to conclude that there are two paralogous copies of FAE1, one on each of the B. napus A and C genomes. • A new procedure is demonstrated to identify novel mutations in situations where two or more very similar paralogous gene copies exist in a genome. The procedure involves TILLING of single plants, using existing SNPs as a positive control, and is able to distinguish novel mutations based on primer pairs designed to amplify both FAE1 paralogues simultaneously. • The procedure was applied to 1344 M2 plants, with 19 mutations identified, of which three were functionally compromised with reduced seed erucic acid content. [ABSTRACT FROM AUTHOR]

We report two new point mutations causing α-thalassemia (α-thal) that could not be characterized by conventional biochemical studies. The first mutation is a single base substitution at codon 123 of the α1-globin gene [α123(H6)Ala→Pro, GCC>CCC (α1)] and leads to the substitution of a proline residue in the H helix. The resulting unstable hemoglobin (Hb) variant has been named Hb Voreppe. The second is a frameshift of the α2 gene due to a deletion (-C), either of the third base of codon 112 or of the first base of codon 113, that causes a premature stop codon at position 132. [ABSTRACT FROM AUTHOR]

Abstract: Duchenne/Becker muscular dystrophy (DMD/BMD), the most common X-linked muscular dystrophy is caused by mutations in the enormously large DMD gene, encoding the protein called dystrophin. This gene was screened in a group of 27 unrelated Bulgarian DMD/BMD patients by MLPA analysis/complete sequencing. We managed to clarify the disease-causing mutation in 96.3% of the analyzed families. The MLPA analysis revealed 17 deletions (including a deletion of the very last exon 79), 6 duplications and 1 point mutation. Two additional point mutations (one of them novel) were detected after complete sequencing of the DMD gene. Altogether, 25 carriers and 11 noncarriers were detected in our families. The MLPA test proved to be a powerful tool in detecting deletions/duplications and in some cases point mutations/polymorphisms along the DMD gene. Using this approach in combination with a direct gene sequencing a number of Bulgarian DMD/BMD patients are genetically clarified and prepared for gene therapy in future. [Copyright &y& Elsevier]

The conformational stability of more than 1500 protein mutants was modelled by a proteometric approach using amino acid sequence autocorrelation vector (AASA) formalism. 48 amino acid/residue properties selected from the AAindex database weighted the AASA vectors. Genetic algorithm-optimised support vector machine (GA-SVM), trained with subset of AASA descriptors, yielded predictive classification and regression models of unfolding Gibbs free energy change (ΔΔG). Function mapping and binary SVM models correctly predicted about 50 and 80% of ΔΔG variances and signs in crossvalidation experiments, respectively. Test set prediction showed adequate accuracies about 70% for stable single and double point mutants. Conformational stability depended on autocorrelations at medium and long ranges in the mutant sequences of general structural, physico-chemical and thermodynamical properties relative to protein hydration process. A preliminary version of the predictor is available online at http://gibk21.bse.kyutech.ac.jp/llamosa/ddG-AASA/ddG_AASA.html. [ABSTRACT FROM AUTHOR]

Abstract: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide including southern China. We developed and validated a reverse dot blot (RDB) assay for the rapid and simultaneous genotyping of six mutations (c.95A>G, c.871G>A, c.1004C>T, c.1024C>T, c.1376G>T and c.1388G>A), that were common mutations in the Chinese G6PD deficiency population, and one polymorphism (c.1311C>T). Reliable genotyping of wild-type and mutant genomic DNA samples was achieved by means of a test strip onto which allele-specific oligonucleotide probe lines are fixed in parallel. This method involves a multiplex PCR amplification of three fragments in the G6PD target sequence and a manual hybridization/detection protocol. The entire procedure starting from blood sampling to the identification of mutations requires less than 6 h. The diagnostic reliability of this reverse dot blot assay was evaluated on 207 pre-typed samples by using direct DNA sequence analysis in a blind study. The reverse dot blot typing was in complete concordance with the reference method. The reverse dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify common G6PD mutations in Chinese population. [Copyright &y& Elsevier]

Abstract: Development of molecular techniques with analytical capability of mutation detection can realize the medical diagnosis of diseases and improve people''s health. β-Thalassemia is one of the most prevalent genetic disorders in Iran and using a simple and rapid test in laboratories for the mass screening and prenatal diagnosis is essential. Here, we described a simple method for rapid detection of four common β-thalassemia point mutations in Iranians (IVS-II-1 (G→A), IVS-I-5 (G→C), FSC 8/9 (+G), IVS-I-110 (G→A)) using a PCR-ELISA genotyping system. After DNA isolation from whole blood, a segment of β-globin gene was amplified by DIG-labeling PCR. The DIG-labeled PCR amplicons were denatured and added to biotinylated normal probe (for normal gene allele) and mutant probe (for mutant gene allele). The hybrids were detected by colorimetric ELISA method. The optical densities obtained using normal and mutant probes with heterozygous PCR products were very similar. The optical densities obtained using mutant probes were higher than normal probes with homozygous PCR products. In vice versa, the optical densities obtained using normal probes were higher than mutant probes with normal PCR products. All the results demonstrated that the PCR-ELISA has similar specificity in comparison to the amplification refractory mutation system. [Copyright &y& Elsevier]

Abstract: Background: Hemophilia A is a common hereditary bleeding disorder caused mainly by mutations in the Factor VIII (FVIII) gene, which results in defective or absent FVIII protein. Most of the causative mutations arise from the germ cells, which leads to either heterozygous or hemizygous state for the mutation in the next generation. Germline or somatic mosaic may result due to a de novo mutation during early embryogenesis. Method: We analyzed 14 families of Indian origin with Hemophilia A [sporadic and severe] for the presence of mosaic individuals by employing Allele Specific PCR, mutation enrichment experiment and sequencing. Result: Nine families had point mutations, 3 families had small deletions or insertions, 2 families had splice site mutations. The origin of the de novo mutation was assigned to the patients'' mother in 8 families. For 4 families it was assigned to the maternal grandmother and to the maternal grandfather in 2 families. In a single family somatic mosaic was detected. Conclusion: The presence of somatic mosaic in families with sporadic Hemophilia A in India may confound risk estimation during genetic counseling. [Copyright &y& Elsevier]

Abstract: We report a sensitive method to detect point mutations in proteins from complex samples. The method is based on surface-enhanced laser desorption/ionization time-of-flight (SELDI–ToF) MS but can be extended to other MS platforms. The target protein in this study is the lysosomal enzyme glucocerebrosidase (GC), the key enzyme in Gaucher’s disease. Deficiency of GC activity results in accumulation of glucosylceramide in macrophages. The relationship between GC genotypes and Gaucher’s patient phenotypes is not strict. The possibility to measure protein levels of GC in clinical samples may provide deeper insight into the phenomenology of Gaucher’s disease. For this purpose, GC was isolated in a single enrichment step through interaction with an immobilized monoclonal antibody, 8E4. After on-chip digestion of the antibody–antigen complex with trypsin, a total of 25 GC peptides were identified (sequence coverage ∼60%), including several peptides containing mutated amino acid residues. The described methodology allows mutational analysis on the protein level, directly measured on complex biological samples without the necessity of elaborate purification procedures. [Copyright &y& Elsevier]

Predicting protein stability changes upon point mutation is important for understanding protein structure and designing new proteins. Autocorrelation vector formalism was extended to amino acid sequences and 3D conformations for encoding protein structural information with modeling purpose. Protein autocorrelation vectors were weighted by 48 amino acid/residue properties selected from the AAindex database. Ensembles of Bayesian-regularized genetic neural networks (BRGNNs) trained with amino acid sequence autocorrelation (AASA) vectors and amino acid 3D autocorrelation (AA3DA) vectors yielded predictive models of the change of unfolding Gibbs free energy change (ΔΔG) of chymotrypsin Inhibitor 2 protein mutants. The ensemble predictor described about 58 and 72% of the data variances in test sets for AASA and AA3DA models, respectively. Optimum sequence and 3D-based ensembles exhibit high effects on relevant structural (volume, solvent-accessible surface area), physico-chemical (hydrophilicity/hydrophobicity-related) and thermodynamic (hydration parameters) properties. [ABSTRACT FROM AUTHOR]

Abstract: Development of novel computational approaches for modeling protein properties is a main goal in applied Proteomics. In this work, we reported the extension of the radial distribution function (RDF) scores formalism to proteins for encoding 3D structural information with modeling purposes. Protein-RDF (P-RDF) scores measure spherical distributions on protein 3D structure of 48 amino acids/residues properties selected from the AAindex data base. P-RDF scores were tested for building predictive models of the change of thermal unfolding Gibbs free energy change (ΔΔG) of chymotrypsin inhibitor 2 upon mutations. In this sense, an ensemble of Bayesian-Regularized Genetic Neural Networks (BRGNNs) yielded an optimum nonlinear model for the conformational stability. The ensemble predictor described about 84% and 70% variance of the data in training and test sets, respectively. [Copyright &y& Elsevier]

Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry’s disease (FD), steroid 21-hydroxylase deficiency (21-HD) and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases, where other techniques have failed. [ABSTRACT FROM AUTHOR]

Euclidean distance counts derived from the protein 2D graphs were used for encoding protein structural information. A total of 35 amino acid 2D distance count (AA2DC) descriptors were calculated from the Euclidean distance matrices (EDM) derived from the 2D graphs at distances ranging from 0.05 to 1.8 units with a lag of 0.05 units. AA2DC descriptors were tested for building predictive classification model of the signs of the change of thermal unfolding Gibbs free energy change (ΔΔG) of a large data set of 2048 single point mutations on 64 proteins. A support vector machine (SVM) classifier with a Radial Basis Function kernel was implemented for classifying the conformational stability of protein mutants. Temperature and pH of the ΔΔG experimental measurements were also conveniently used for SVM training in addition to calculated AA2DC descriptors. The optimum SVM model correctly predicted about 72% of ΔΔG signs in crossvalidation test for all the dataset and also for stable and unstable mutant separately. To the best of our knowledge, this level of accuracy for stable mutant recognition is the highest ever reported for a predictor using sequence information. Furthermore, the classifier adequately recognized unstable mutants of human prion protein and human transthyretin associated to diseases. [ABSTRACT FROM AUTHOR]

Abstract: PrP has a central role in the Transmissible Spongiform Encephalopathies (TSEs), and mutations and polymorphisms in host PrP can profoundly alter the host''s susceptibility to a TSE agent. However, precisely how host PrP influences the outcome of disease has not been established. To investigate this we have produced by gene targeting a series of inbred lines of transgenic mice expressing different PrP genes. This allows us to study directly the influence of the host PrP gene in TSEs. We have examined the role of glycosylation, point mutations, polymorphisms and PrP from different species on host susceptibility and the disease process both within the murine species and across species barriers. [Copyright &y& Elsevier]

The discovery of natural RNA sensors that respond to a change in the environment by a conformational switch can be utilized for various biotechnological and nanobiotechnological advances. One class of RNA sensors is the riboswitch: an RNA genetic control element that is capable of sensing small molecules, responding to a deviation in ligand concentration with a structural change. Riboswitches are modularly built from smaller components. Computational methods can potentially be utilized in assembling these building block components and offering improvements in the biochemical design process. We describe a computational procedure to design RNA switches from building blocks with favorable properties. To achieve maximal throughput for genetic control purposes, future designer RNA switches can be assembled based on a computerized preprocessing buildup of the constituent domains, namely the aptamer and the expression platform in the case of a synthetic riboswitch. Conformational switching is enabled by the RNA versatility to possess two highly stable states that are energetically close to each other but topologically distinct, separated by an energy barrier between them. Initially, computer simulations can produce a list of short sequences that switch between two conformers when trigerred by point mutations or temperature. The short sequences should possess an additional desirable property; when these selected small RNA switch segments are attached to various aptamers, the ligand binding mechanism should replace the aforementioned event triggers, which will no longer be effective for crossing the energy barrier. In the assembled RNA sequence, energy minimization folding predictions should then show no difference between the folded structure of the entire sequence relative to the folded structure of each of its constituents. Moreover, energy minimization methods applied on the entire sequence could aid at this preprocessing stage by exhibiting high mutational robustness to- - capture the stability of the formed hairpin in the expression platform. The above computer-assisted assembly procedure together with application specific considerations may further be tailored for therapeutic gene regulation [ABSTRACT FROM PUBLISHER]

Abstract: Several proteins, including transthyretin (TTR), can generate in tissues extracellular insoluble aggregates, in the form of fibrils, that are associated with pathological states known as amyloidoses. To date, more than 80 different TTR point mutations have been associated with hereditary amyloidosis in humans. In vitro, the formation of amyloid fibrils by human TTR is known to be triggered by acidic pH. We show here that, in vitro, the natural amyloidogenic I84S and the non-natural I84A TTR mutant forms exhibit a propensity to produce fibrils in an acidic medium significantly higher than that of wild-type TTR. The two mutant forms have been crystallized at both neutral and acidic pH. Their neutral pH crystal structures are very similar to that of wild-type TTR, consistent with previous evidence indicating that only minor structural changes are induced by amyloidogenic mutations. On the contrary, their crystal structures at moderately low pH (4.6) show significant conformational differences as compared to their neutral pH structures. Remarkably, such changes are not induced in wild-type TTR crystallized at low pH. The most relevant consist of the unwinding of the TTR short α-helix and of the change in conformation of the loop connecting the α-helix to β-strand F. Only one monomer of the crystallographic dimer is affected, causing a disruption of the tetrameric symmetry. This asymmetry and a possible destabilization of the tetrameric quaternary structure of TTR may be responsible for the amyloidogenic potential of the two TTR mutant forms at low pH. [Copyright &y& Elsevier]

Fructans are fructose polymers that are synthesized from sucrose by fructosyltransferases. Fructosyltransferases are present in unrelated plant families suggesting a polyphyletic origin for their transglycosylation activity. Based on sequence comparisons and enzymatic properties, fructosyltransferases are proposed to have evolved from vacuolar invertases. Between 1% and 5% of the total activity of vacuolar invertase is transglycosylating activity. We investigated the nature of the changes that can convert a hydrolysing invertase into a transglycosylating enzyme. Remarkably, replacing 33 amino acids (amino acids 143-175) corresponding to the N-terminus of the mature onion vacuolar invertase with the corresponding region of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) led to a shift in activity from hydrolysis of sucrose towards transglycosylation between two sucrose molecules. The substituted N-terminal region contains the sucrose-binding box that harbours the nucleophile involved in sucrose hydrolysis (Asp 164). Subsequent research into the individual amino acids responsible for the enhanced transglycosylation activity revealed that mutations in amino acids Trp 161 and Asn 166, can give rise to a shift towards polymerase activity. Changing the amino acid at either of these positions in the sucrose-binding box increases the transglycosylation capacity of invertases two- to threefold compared to wild type. Combining the two mutations had an additive effect on transglycosylation ability, resulting in an approximately fourfold enhancement. The mutations generated correspond with natural variation present in the sucrose-binding boxes of vacuolar invertases and fructosyltransferases. These relatively small changes that increase the transglycosylation capacity of invertases might explain the polyphyletic origin of the fructan accumulation trait. [ABSTRACT FROM AUTHOR]

Abstract: To examine the resistance pattern and point mutations present in the carbamate-resistant strain of housefly (SH-CBR), Musca domestica, from Shanghai, China, the fly’s resistance spectrum and k i ratios to organophosphate (OP) and carbamate (CB) insecticides were determined. The cDNA encoding acetylcholinesterase (AChE) from both a susceptible control strain (SH-S) and the SH-CBR strain of housefly were cloned and sequenced using RT-PCR. Based on two major patterns of target site resistance suggested by Russell et al. [R. J. Russell, C. Claudianos, P. M. Campbell, I. Horne, T. D. Sutherland, J. G. Oakeshott, Two major classes of target site insensitivity mutations confer resistance to organophosphate and carbamate, Pestic. Biochem. Physiol. 79 (2004), 84–93.], the present results indicate a Pattern I resistance: resistance to CBs was greater than that to OPs; the SH-S/SH-CBR k i ratios were also greater for CBs than for OPs. The cDNA was 2079 nucleotides long, encoding a protein of 693 amino acids. The cDNA deduced amino acid sequence of the housefly had a high degree of identity with previously described insect AChEs and exhibited the same structural feature as vertebrate TcAChE. Three mutations in the region of the active site, V261L, G343A, and F408Y, which were identical to those identified in the 77M and YBOL housefly strains, were identified in the AChE from the SH-CBR strain. Additionally, a novel mutation, D422L, was found at the outer surface of the protein where the residue presumably could not interact directly with amino acid residues lining the active site gorge. Homologous modelling of housefly AChE, based on the high resolution crystal structure of fruit fly (Drosophila melanogaster) AChE, indicated that D422 may be involved in a salt bridge with H341. The distance between D422 and H341 is predicted to be 3.8Å, and modification of the charge on the side chain of D422 (D to V) would likely affect the profile of the acyl pocket via the Y339 component. Such a structural change in the acyl pocket in the mutant may underlie the decreased affinity of AChE for CBs. [Copyright &y& Elsevier]

We report three new α-thalassemia (thal) point mutations detected during newborn screening for hemoglobinopathies. The first mutation is a single nucleotide deletion (-A) that abolishes the translation initiation codon of the α2-globin gene, detected in a newborn of Hmong ethnicity who carried the Southeast Asian α°-thal deletion αTα/- -SEA) The second mutation, a frameshift caused by a single nucleotide deletion in exon 2 of the α1-globin gene [codon 78 (-C)], was detected in a Black/ Chinese newborn who also carried the Southeast Asian α°-thal deletion (ααT/- -SEA). The third mutation was a frameshift in exon 3 of the α2-globin gene, codons 113/114 (-C). This mutation was detected in a newborn who carried the 3. 7 kb α+-thall deletion (αTα/-α3.7). [ABSTRACT FROM AUTHOR]

Abstract: Dihydropteroate synthase gene of Plasmodium vivax was recently identified. In the present study, the sequences of the dyhydropteroate synthase gene of 68 P. vivax isolates from various geographic areas were compared. Sequencing revealed limited polymorphism at codons 383 and 553 in all analyzed samples. Interstrain analysis showed several genotypic variations in the tandem repeats domain which produce length polymorphism between different parasite isolates. [Copyright &y& Elsevier]

Abstract: Oncogenic mutations of the receptor tyrosine kinase KIT are encountered in myeloid leukemia and various solid tumors, including gastrointestinal stromal tumors. We previously identified the human oncogenic germ line mutant KITK642E, a substitution in the tyrosine kinase 1 domain (TK1D) in a familial form of gastrointestinal stromal tumors. The effects of oncogenic KIT mutants on cell signaling and regulation are complex. Cellular models are valuable basic tools to tailor novel strategies on specific cellular and molecular bases for tumors expressing KIT oncogenic mutants. Murine KITWT and the murine homologues of human KIT oncogenic mutants, further referred to as KITK641E and KITdel559, a point deletion in the juxtamembrane domain (JMD), were stably expressed in IL-3-dependent Ba/F3 cells. Major differences in the constitutively activation of Akt/PKB, MAP kinases and STATs pathways were observed between KITK641E and KITdel559, whereas KIT ligand elicited responses in both mutants. Noteworthy, the protein level of the phosphoinositide phosphatase SHIP1, but not SHIP2 and PTEN, was reduced in KITK641E only while inhibition of KIT phosphorylation reversibly raised SHIP1 level in both JMD and TK1D oncogenic mutants, unraveling the control of SHIP protein level by KIT phosphorylation. [Copyright &y& Elsevier]

Abstract: The BERTS strain of Colorado potato beetle (CPB) was found to be highly resistant to N-methyl carbofuran but relatively susceptible to azinphosmethyl. N-Methyl carbofuran resistance was found to correlate well with acetylcholinesterase (AChE) insensitivity. In becoming resistant to N-methyl carbofuran, the AChE of the BERTS strain became more sensitive to N-propyl carbofuran inhibition. This negative cross-insensitivity correlated well to the increased relative toxicity of the BERTS strain to N-propyl carbofuran compared to the susceptible SS strain. BERTS beetles were sorted into BERTS-R and BERTS-S substrains using their AChE activity profiles. Sequence comparisons of AChE cDNAs from the two substrains revealed the presence of the point mutation that results in the S291G substitution previously found in the AChE of the azinphosmethyl-resistant AZ-R strain of CPB. A novel mutation present only in BERTS-S CPB, however, resulted in an additional I392T substitution in the AChE and apparently reverses the resistance conferring properties of the S291G substitution. [Copyright &y& Elsevier]

Abstract: Mutations in Dihydrofolate Reductase (dhfr) gene of Plasmodium vivax are known to be associated with resistance to antifolate drugs. To analyze the extent of these mutations in P. vivax population in India, dhfr gene was isolated and sequenced for 121 P. vivax isolates originating from different geographical regions of Indian subcontinent. These sequences were compared with the gene sequence that represent wild type sequence (accession no. X98123). P. vivax dhfr (Pvdhfr) sequences showed limited polymorphism and about 70% isolates showed wild type dhfr sequence. A total of 36 mutations were found at 11 positions in 121 isolates. A majority of mutant isolates showed double mutations at residues 58 (S→R) and 117 (S→N), known to be associated with pyrimethamine resistance, but only 19% showed double mutations at residues 57 (F→L) and 58 (S→R). Pvdhfr alleles showing quadruple mutation (F57L, S58R, T61M andS117T) were found in two isolates. Three other mutations reported earlier at residue 13, 33 and 173 were not found in any of the Isolates. Six novel mutations at residues 38 (R→G), 93 (S→C), 109 (S→H), 131 (R→G), 159 (V→A) and 188 (I→V) were observed in seven isolates. Whether these novel mutations are linked to pyrimethamine resistance remains to be established. [Copyright &y& Elsevier]

BRCA1 mutations are associated with a higher risk of breast (BC) and ovarian cancer in women. Testing for such mutations allows BC prognosis, selection of an individual treatment strategy, and prevention of disease recurrence. Hybridization on a hydrogel microarray was developed for identifying point mutations in BRCA1. The microarray was designed to detect five-point mutations: 185delAG, 300T→G, 4153delA, 4158A→G, and 5382insC. The microarray was tested with 36 control specimens with known genotypes and used to examine 65 BC patients. The results demonstrated the advantage of employing the microarray in analyzing BRCA1 mutations. [ABSTRACT FROM AUTHOR]