Adel Pezeshki

Impurity profiling quality control testing of synthetic peptides using liquid chromatography-photodiode array-fluorescence and liquid chromatography-electrospray ionization-mass spectrometry: the obestatin case.

Following several conflicting publications, the inability to reproduce the original findings on in vitro obestatin binding and activation of GPR39 receptors was recently reported by its discoverers, and several hypotheses to rationalize these findings were proposed. Based on one of these postulations (i.e., presence of impurities), peptide identity and impurity profiles were thoroughly evaluated on obestatin peptides obtained from five different manufacturers, as used by the different research groups. We found that one of the products examined was in reality a totally different peptide and that the quality of two-thirds of the other peptides was insufficient for in vitro and in vivo experiments (i.e., peptide purity less than 95% and/or individual impurities exceeding 1%). These observations question the divergent conclusions reported in the literature about the activity of obestatin. Therefore, we strongly recommend appropriate quality control testing before using any peptides for biomedical research purposes.

Hydrophilic interaction LC of peptides: Columns comparison and clustering

A wide variety of hydrophilic interaction chromatography (HILIC) stationary phase surface chemistries are currently available. Although their selectivity can be considerably different, column comparison or clustering using peptides is limited. In this study, ten pharmaceutically relevant model peptides are analyzed on seven different HILIC columns (bare silica, amide, poly-hydroxyethyl aspartamide, diol and zwitterionic) for the evaluation of their performance and classification. The responses examined include single and multiple responses: plate number, asymmetry factor, LOD, geometric mean resolution, resolution product, time corrected resolution product, peak capacity and chromatographic response function. Column classification was performed using hierarchical clustering and principal component analysis. Moreover, the overall performance quality of the HILIC columns was compared using a linear desirability function. Hierarchical clustering and principal component analysis showed consistent clusters. The zwitterionic phase was clustered apart from the other HILIC columns and both poly-aspartamide columns were clustered together. In addition, the two bare silica phases represent two different clusters, and thus different selectivities. Overall, the responses showed the best performance for one of the bare silica columns (Alltima-Alltech), followed by the zwitterionic phase (ZIC)-HILIC. Thus, these columns, belonging to different clusters, were found to be the best performing systems in pharmaceutical peptide analysis for the selected peptide set.

Effects of continuous lactation and short dry periods on mammary function and animal health

The dry period is required to facilitate cell turnover in the bovine mammary gland in order to optimize milk yield in the next lactation. Traditionally, an 8-week dry period has been a standard management practice for dairy cows based on retrospective analyses of milk yields following various dry period lengths. However, as milk production per cow has increased, transitioning cows from the nonlactating state to peak milk yield has grown more problematic. This has prompted new studies on dry period requirements for dairy cows. These studies indicate a clear parity effect on dry period requirement. First parity animals require a 60-day dry period, whereas lactations following later parities demonstrate no negative impact with 30-day dry period or even eliminating the dry period when somatotropin (ST) is also used to maintain milk yields. Shortened dry periods in first parity animals were associated with reduced mammary cell turnover during the dry period and early lactation and increased numbers of senescent cells and reduced functionality of lactating alveolar mammary cells postpartum. Use of ST and increased milking frequency postpartum reduced the impact of shortened dry periods. The majority of new intramammary infections occur during the dry period and persist into the following lactation. There is therefore the possibility of altering mastitis incidence by modifying or eliminating the dry period in older parity animals. As the composition of mammary secretions including immunoglobulins may be reduced when the dry period is reduced or eliminated, there is the possibility that the immune status of cows during the peripartum period is influenced by the length of the dry period.

Adsorption of peptides at the sample drying step : Influence of solvent evaporation technique, vial material and solution additive

Although the efficient and careful removal of solvent from samples by centrifugal evaporation or freeze-drying methods is an important step in peptidomics, the recovery of peptides has not yet been fully investigated with these sample drying methods. Moreover, the surface adsorption of the peptides by the container and efforts to reduce this adsorption by organic additives is only scarcely elaborated until now. In this experiment, the recovery of five model peptides, i.e. bovine insulin, mouse obestatin, goserelin, buserelin and leucine-enkephalin was investigated applying dimethylsulfoxide (DMSO), dimethylformamide (DMF), polyethylene glycol 400 (PEG 400), mannitol and n-nonyl-beta-d-glucopyranoside (C(9)-Glu) in function of the two applied solvent evaporation processes (freeze-drying vs. centrifugal evaporation) and vial types, i.e. polypropylene (PP) and glass. Under our experimental conditions, drying resulted in a decreased recovery of the model peptides by 10% on average. Insulin showed the lowest recovery value relative to the other model peptides. For both drying methods, recovery of the model peptides was increased when C(9)-Glu was present. Overall, the use of PP vials is proposed for freeze-drying, while glass vials are found to be more suitable for centrifugal evaporation. The presence of PEG 400 in PP vials caused significantly reduced recoveries for all model peptides using centrifugal evaporation, although this was not observed in glass vials. As a general conclusion, applying C(9)-Glu as an additive along with choosing appropriate vial type (i.e. PP for lyophilization and glass for centrifugal evaporation) can avoid or diminish peptide loss during the evaporation procedure.

The effect of dry period length reduction to 28 days on the performance of multiparous dairy cows in the subsequent lactation

In a controlled study, the effects of shortened and conventional dry periods (28.2 ± 4.6 vs. 48.8 ± 4 d) on performance of multiparous cow were evaluated. Seventy multiparous Holstein cows were randomly assigned to either a 28- or a 49-d dry period length (DPL). Nine cows were removed from the study and 61 cows were used in the statistical analysis. Cows in the 28-d DPL group were moved to close-up pen and fed only close-up ration during the dry period, whereas those in the 49-d DPL group were given a far-off diet after dry-off until 28 d before expected calving, and then fed the same diet as the cows in the 28-d DPL group. Daily milk yield production was significantly reduced in the 28-d DPL group compared with the 49-d DPL group through 210 d in milk (DIM). Neither milk protein nor milk fat was changed in experimental groups. However, fat yield tended to be greater in the 49 d DPL compared with the 28 d DPL. Cows with 28 d DPL lost less body condition in peripartum and gained more body condition score (...

Differential gene expression of the toll-like receptor-4 cascade and neutrophil function in early- and mid-lactating dairy cows

During early lactation, neutrophils display several reduced immune functions. Particularly, a delayed recruitment of neutrophils into the infected udder seems to be one of the underlying events involved in the severity of postpartum Escherichia coli intramammary infections. The purpose of this study was to analyze the effect of in vitro chemotaxis and diapedesis on the expression of toll-like receptor-4 (TLR4)-related genes in bovine blood neutrophils isolated from 10 early-lactating (EL) and 10 mid-lactating (ML) cows. Functional characterization of the neutrophil population was performed by measuring phagocytosis and production of reactive oxygen species (chemiluminescence). Messenger RNA was extracted from neutrophils, and the expression of TLR4 and associated genes in EL and ML cows was analyzed by reverse-transcription quantitative PCR. To study the effect of chemotaxis and diapedesis on the expression of genes of the TLR4 cascade, neutrophils were stimulated to (trans)migrate in response to C5a using in vitro models. Our salient findings were that both neutrophil migration in vitro and lactation stage induced significant changes in the expression of several genes of the TLR4 signaling cascade. Before migration, expression of TRAF6, ATF3, RELA, IL8, and C5aR were lower in EL than in ML cows. Diapedesis and chemotaxis induced an increase in expression of TLR4, ATF3, and IL8 in both EL and ML cows. Diapedesis resulted in a downregulation of Syk, a TLR4-associated gene, in ML cows. This study shows that the perturbations in neutrophil functions during EL are accompanied by modulation of TLR4 pathway genes. These data can contribute to the understanding of the mechanisms explaining the relationship between stage of lactation and risk of severe E. coli mastitis.

Variation of inflammatory dynamics and mediators in primiparous cows after intramammary challenge with Escherichia coli.

The objective of the current study was to investigate (i) the outcome of experimentally induced Escherichia coli mastitis in primiparous cows during early lactation in relation with production of eicosanoids and inflammatory indicators, and (ii) the validity of thermography to evaluate temperature changes on udder skin surface after experimentally induced E. coli mastitis. Nine primiparous Holstein Friesian cows were inoculated 24 ± 6 days (d) after parturition in both left quarters with E. coli P4 serotype O32:H37. Blood and milk samples were collected before and after challenge with E. coli. The infrared images were taken from the caudal view of the udder following challenge with E. coli. No relationship was detected between severity of mastitis and changes of thromboxane B2 (TXB2), leukotriene B4 (LTB4) and lipoxin A4 (LXA4). However, prostaglandin E2 (PGE2) was related to systemic disease severity during E. coli mastitis. Moreover, reduced somatic cell count (SCC), fewer circulating basophils, increased concentration of tumor necrosis factor-α (TNF-α) and higher milk sodium and lower milk potassium concentrations were related to systemic disease severity. The thermal camera was capable of detecting 2-3°C temperature changes on udder skin surface of cows inoculated with E. coli. Peak of udder skin temperature occurred after peak of rectal temperature and appearance of local signs of induced E. coli mastitis. Although infrared thermography was a successful method for detecting the changes in udder skin surface temperature following intramammary challenge with E. coli, it did not show to be a promising tool for early detection of mastitis.

An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence

The dry period is necessary to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation. An 8-week dry period has long been the golden standard of management for dairy cows. Genetic improvements and new management technologies have led to higher milk production and a need for re-evaluation of the dry period length. Over the last decade, dry period length has been proposed to be shortened or eliminated mainly from an economic point of view. However, the influence of modified dry period length on the immune defence of the bovine mammary gland and the occurrence of new intramammary infections has not yet been appreciated. The objective of this review is to discuss the bovine mammary gland biology, defence and systemic health when the dry period length is modified. Shortening or eliminating the dry period may minimize or remove the impact of milk accumulation at dry off, thereby lessening the immunodeficiency of the dam that is characteristic of this period. Composition of mammary secretions may change and the extent of tissue remodelling may be reduced when the dry period is reduced or eliminated. Additionally, impact of the dry period length on energy and nutritional status, and on hormonal and local regulatory factors, lead us to hypothesize that changing the dry period length might also affect the response to intramammary infection. It is concluded that there is a need to integrate mammary gland biology and defence mechanisms in studies dealing with modified dry period lengths.

REVIEW ARTICLE: An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence

The dry period is necessary to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation. An 8-week dry period has long been the golden standard of management for dairy cows. Genetic improvements and new management technologies have led to higher milk production and a need for re-evaluation of the dry period length. Over the last decade, dry period length has been proposed to be shortened or eliminated mainly from an economic point of view. However, the influence of modified dry period length on the immune defence of the bovine mammary gland and the occurrence of new intramammary infections has not yet been appreciated. The objective of this review is to discuss the bovine mammary gland biology, defence and systemic health when the dry period length is modified. Shortening or eliminating the dry period may minimize or remove the impact of milk accumulation at dry off, thereby lessening the immunodeficiency of the dam that is characteristic of this period. Composition of mammary secretions may change and the extent of tissue remodelling may be reduced when the dry period is reduced or eliminated. Additionally, impact of the dry period length on energy and nutritional status, and on hormonal and local regulatory factors, lead us to hypothesize that changing the dry period length might also affect the response to intramammary infection. It is concluded that there is a need to integrate mammary gland biology and defence mechanisms in studies dealing with modified dry period lengths.

REVIEW ARTICLE: An integrated view on how the management of the dry period length of lactating cows could affect mammary biology and defence: Dry period length and bovine mammary defence

The dry period is necessary to facilitate cell turnover in the bovine mammary gland and to optimize milk production in the next lactation. An 8-week dry period has long been the golden standard of management for dairy cows. Genetic improvements and new management technologies have led to higher milk production and a need for re-evaluation of the dry period length. Over the last decade, dry period length has been proposed to be shortened or eliminated mainly from an economic point of view. However, the influence of modified dry period length on the immune defence of the bovine mammary gland and the occurrence of new intramammary infections has not yet been appreciated. The objective of this review is to discuss the bovine mammary gland biology, defence and systemic health when the dry period length is modified. Shortening or eliminating the dry period may minimize or remove the impact of milk accumulation at dry off, thereby lessening the immunodeficiency of the dam that is characteristic of this period. Composition of mammary secretions may change and the extent of tissue remodelling may be reduced when the dry period is reduced or eliminated. Additionally, impact of the dry period length on energy and nutritional status, and on hormonal and local regulatory factors, lead us to hypothesize that changing the dry period length might also affect the response to intramammary infection. It is concluded that there is a need to integrate mammary gland biology and defence mechanisms in studies dealing with modified dry period lengths.