Application of one-step liquid chromatography–electrospray tandem MS/MS and collision-induced dissociation to quantification of ezetimibe and identification of its glucuronated metabolite in human serum: A pharmacokinetic study

A new one-step liquid chromatography–electrospray tandem MS/MS method is described to quantify ezetimibe (EZM) a novel lipid lowering drug in human serum. Also using collision-induced dissociation (CID) of the analyte, identification and chromatographic separation of its major metabolite, ezetimibe glucuronide (EZM-G) is achieved in this study. A thawed serum aliquot of 100 μL was deproteinated by addition of 500 μL methanol containing omeprazole as internal standard (I.S.). Separation of the drug, its metabolite and the I.S. were achieved using acetonitrile–water (70:30, v/v) as mobile phase at flow rate of 0.5 mL/min on a MZ PerfectSil target C18 column. Multiple reaction monitoring (MRM) mode of precursor–product ion transition (408.7 → 272.0 for EZM and 345 → 194.5 for the I.S.) was applied for detection and quantification of the drug while, EZM-G was chromatographically separated and identified using CID. The analytical method was linear over the concentration range of 1–32 ng/mL of EZM in human serum with a limit of quantification of 1 ng/mL. The coefficient variation values of both inter- and intra-day analysis were less than 8% whereas the percentage error was less than 3.7. The validated method was applied in a randomized cross-over bioequivalence study of two different EZM preparations in 24 healthy volunteers.     

Crystal structures of MMP-9 complexes with five inhibitors: contribution of the flexible Arg424 side-chain to selectivity

Human matrix metalloproteinase 9 (MMP-9), also called gelatinase B, is particularly involved in inflammatory processes, bone remodelling and wound healing, but is also implicated in pathological processes such as rheumatoid arthritis, atherosclerosis, tumour growth, and metastasis. We have prepared the inactive E402Q mutant of the truncated catalytic domain of human MMP-9 and co-crystallized it with active site-directed synthetic inhibitors of different binding types. Here, we present the X-ray structures of five MMP-9 complexes with gelatinase-specific, tight binding inhibitors: a phosphinic acid (AM-409), a pyrimidine-2,4,6-trione (RO-206-0222), two carboxylate (An-1 and MJ-24), and a trifluoromethyl hydroxamic acid inhibitor (MS-560). These compounds bind by making a compromise between optimal coordination of the catalytic zinc, favourable hydrogen bond formation in the active-site cleft, and accommodation of their large hydrophobic P1' groups in the slightly flexible S1' cavity, which exhibits distinct rotational conformations of the Pro421 carbonyl group in each complex. In all these structures, the side-chain of Arg424 located at the bottom of the S1' cavity is not defined in the electron density beyond C(gamma), indicating its mobility. However, we suggest that the mobile Arg424 side-chain partially blocks the S1' cavity, which might explain the weaker binding of most inhibitors with a long P1' side-chain for MMP-9 compared with the closely related MMP-2 (gelatinase A), which exhibits a short threonine side-chain at the equivalent position. These novel structural details should facilitate the design of more selective MMP-9 inhibitors.     

Thermal requirement for development of Sancassania rodionovi (Acari: Acaridae) on mushrooms

The free-living mite species Sancassania rodionovi (Zachvatkin) (Acari: Acaridae), is a serious pest of mushrooms in Iran. Studies were conducted to examine the development of this mite in relation to temperature on two mushroom species: Agaricus bisporus Lange (button mushroom) and Pleurotus ostreatus Kummer (oyster mushroom). The developmental time of this acarid mite was studied at eight constant temperatures, ranging from 5 to 40 degrees C, and developmental rates were modeled as a function of temperature. Sancassania rodionovi completed immature development in 17.35 +/- 0.58 and 20.17 +/- 0.88 d at 25 degrees C on button and oyster mushrooms, respectively. When the mite fed on button mushroom, the rate of development increased gradually from 10 to 35 degrees C. Using a linear model, the developmental zero was estimated to be 3.50 degrees C with a thermal constant of 357.14 degree-days. The Logan 10, Briere 1, and Thermodynamic models adequately described the data for this mite and yielded R2 values >0.95; these models provided estimates of optimum temperature for development of 33.244, 32.145, and 32.148 degrees C, respectively. Understanding the influence of temperature on development of S. rodionovi is discussed with respect to pest management in mushroom production.     

Frequency of the 35delG allele causing nonsyndromic recessive deafness in the Algerian patients

Deafness is a heterogeneous disorder showing different patterns of inheritance and involving a multitude of different genes. Mutations in the GJB2 gene encoding connexin 26 (Cx26) protein are a major cause for non-syndromic autosomal recessive and sporadic deafness. Among these mutations, the c.35delG deletion is the most common mutation for sensorineural deafness. One hundred sixteen persons from fifty-eight families were tested by the method based on the principle of PCR-mediated-site-directed mutagenesis (PSDM), followed by a Bsl1 digestion. Mutation c.35delG was diagnosed in sixteen families (11 homozygotes and 5 heterozygotes). The low allelic frequency (17.24%) and low ratio of individuals homozygous (13.8%) and heterozygous (6.9%) for the c.35delG mutation suggest that there are other mutations in the GJB2 gene or other genes responsible for deafness in the Algerian population. This study reports a significant association (P=0.003) between first cousin consanguinity and non-syndromic prelingual deafness.     

The essential peptidoglycan glycosyltransferase MurG forms a complex with proteins involved in lateral envelope growth as well as with proteins involved in cell division in Escherichia coli

In Escherichia coli many enzymes including MurG are directly involved in the synthesis and assembly of peptidoglycan. MurG is an essential glycosyltransferase catalysing the last intracellular step of peptidoglycan synthesis. To elucidate its role during elongation and division events, localization of MurG using immunofluorescence microscopy was performed. MurG exhibited a random distribution in the cell envelope with a relatively higher intensity at the division site. This mid-cell localization was dependent on the presence of a mature divisome. Its localization in the lateral cell wall appeared to require the presence of MreCD. This could be indicative of a potential interaction between MurG and other proteins. Investigating this by immunoprecipitation revealed the association of MurG with MreB and MraY in the same protein complex. In view of this, the loss of rod shape of DeltamreBCD strain could be ascribed to the loss of MurG membrane localization. Consequently, this could prevent the localized supply of the lipid II precursor to the peptidoglycan synthesizing machinery involved in cell elongation. It is postulated that the involvement of MurG in the peptidoglycan synthesis concurs with two complexes, one implicated in cell elongation and the other in division. A model representing the first complex is proposed.     

Analysis of molecular inversion probe performance for allele copy number determination

We have developed a new protocol for using molecular inversion probes to accurately and specifically measure allele copy number. The new protocol provides for significant improvements, including the reduction of input DNA (from 2 μg) by more than 25-fold (to 75 ng total genomic DNA), higher overall precision resulting in one order of magnitude lower false positive rate, and greater dynamic range with accurate absolute copy number up to 60 copies.     

A new report of pre-harvest ear rot of corn caused by Geotrichum candidum from Iran

In summer of 2004, samples of husk looseness ear of corn (Zea mays) (cv. 700-Karaj) were collected from corn fields in Ali-Abad (Jiroft region), Kerman province, Southeastern Iran, for diagnosis of an unusual ear decay. A fungus was isolated from the rotting kernels and subsequently identified as Geotrichum candidum. The fungal pathogen was found to be closely related to G. citri-aurantii (citrus race) based on morphological, physiological and pathogenicity properties. The fungal pathogenicity test was demonstrated by fulfilling Koch's postulates. The pathogen caused rot disease on husk looseness corn kernels in soft-dough stage of ripening. The fungus was also pathogenic on ripe lemon and green and ripe tomato fruits. Fungal isolates of corn were compared to isolates from soft-rotten potato tubers. These two groups of isolates were highly similar on the basis of their morphological, biochemical and pathogenicity characteristics. To our knowledge, this is the first known report of corn ear rot caused by G. candidum in the world.     

Demographic parameters of wrinkling aphid of pistachio leaf, Forda hirsuta Mordv. (Hem.:Pemphigidae) insitu of Rafsanjan's orchards

Wrinkling aphid of pistachio leaf, Forda hirsuta Mordv. (Hem.:Pemphigidae) is one of the pests of pistachio trees. This aphid caused the shrinkage, thickening, and changing the color of the pistachio leaves. Since insect feeds from leave edges, the thick and rolled upward wrinkles were formed, which its green color turned into red. Therefore, its economical damages are out of direct feeding from plant extraction, twisting pistachio leaves, and the decrease of photosynthesis. In this research two orchards and 10 trees that each of them were selected in Rafsanjan region and 58 fundatrix galls, 120 nymphal galls were marked and the demographic parameters for apterus parthenogenesis female of this aphid were calculated via daily observations. The results indicated that intrinsic rates of increase (r) for 1, 2 and 3 generations were 0.01, 0.0638 and 0.0575 femal/femal/days respectively, Doubling time (DT) were 69.31, 10.52 and 12.04 days, respectively, net fecundity rates were 1.71, 11.5 and 7.37 femal/femal/days, respectively, Net fertility rates were 1.11, 8.87 and 5.01 days, respectively, and mean generation times (Tc) were calculated to be 31.5, 32.2 and 31.87 days, respectively. Other reproductive parameters such as gross hatch rate, gross fecundity rate, gross fertility rate, mean age gross fecundity and fertility, mean age net fecundity and fertility, mean age hatch, finite rate of increase (lambda), intrinsic birth rate (b) intrinsic death rate (d) and daily reproductive rate were also calculated. The results revealed that population parameters especially r in the first and second generations were lowest and highest, respectively and mean longevity of fundatrix was 30 days and the born nymphs made separate galls in the edge of pistachio leaves.     

Effects of Stress Modifier Biostimulants on Vegetative Growth,Nutrients, and Antioxidants Contents of Garden Thyme (Thymus vulgaris L.) Under Water Deficit Conditions

The aim of this study is to investigate the effect of stress modulators on vegetative growth, antioxidants, and nutrient content of Thymus vulgaris L. under water deficit stress conditions. A factorial experiment was performed in the form of a randomized complete block design with 10 treatments and 3 replications in the 2019–2020 growing season. The factors were stress modulators at 5 levels (ZN: zinc nano-fertilizer, AA: amino acid, SW: seaweed, HA: humic acid and C: control) and irrigation regime at 2 levels [FIrr: full irrigation (100% field capacity) and DIrr: deficit irrigation (50% field capacity)]. The highest plant height, number of branches, and total dry weight of the garden thyme plant were observed in the foliar application of HA and SW under full irrigation conditions. Relative water content, chlorophyll a and b, and uptake of nutrients (N, P, and K) were reduced under water deficit stress, but the foliar application of stress modulators increased relative water content, chlorophyll content, and nutrient uptake of the garden thyme plant significantly compared with control. The water deficit increased proline content, total flavonoid, and phenol content in the garden thyme plant. So, the highest total flavonoid and phenol content was obtained from plants treated with HA, whereas proline content was higher in the control plants. Soluble sugars and essential oil increased significantly under water deficit stress conditions. The foliar application of HA compared to the control plant increased soluble sugars and essential oil in garden thymes. The activities of catalase, superoxide dismutase, and ascorbate peroxidase enzymes were improved in stress modulator treatments such as HA and SW compared to control plants under water deficit stress conditions. The plants of garden thymes showed a good response to stress modulator treatments under water stress conditions, and HA and SW treatments were found to be more effective.