Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.     

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S‐phase arrest and apoptosis

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27‐mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27‐overexpressing cells displayed an early S‐phase arrest subsequently followed by a strongly increased sub‐G1 fraction. Apoptosis was characterized by PARP‐, CASPASE 3‐, CASPASE 8‐, CASPASE 9‐ and BIM‐ activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock‐induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27‐overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor‐targeting agents, suggesting another pro‐apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well‐established anti‐apoptotic properties of HSP27 in cancer, our study reveals novel pro‐apoptotic functions of HSP27—mediated through both the intrinsic and the extrinsic apoptotic pathways—at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients.     

Stress “Deafness” Reveals Absence of Lexical Marking of Stress or Tone in the Adult Grammar

A Sequence Recall Task with disyllabic stimuli contrasting either for the location of prosodic prominence or for the medial consonant was administered to 150 subjects equally divided over five language groups. Scores showed a significant interaction between type of contrast and language group, such that groups did not differ on their performance on the consonant contrast, while two language groups, Dutch and Japanese, significantly outperformed the three other language groups (French, Indonesian and Persian) on the prosodic contrast. Since only Dutch and Japanese words have unpredictable stress or accent locations, the results are interpreted to mean that stress “deafness” is a property of speakers of languages without lexical stress or tone markings, as opposed to the presence of stress or accent contrasts in phrasal (post-lexical) constructions. Moreover, the degree of transparency between the locations of stress/tone and word boundaries did not appear to affect our results, despite earlier claims that this should have an effect. This finding is of significance for speech processing, language acquisition and phonological theory.     

International External Quality Assessment Study for Molecular Detection of Lassa Virus

Lassa virus (LASV) is a causative agent of hemorrhagic fever in West Africa. In recent years, it has been imported several times to Europe and North America. The method of choice for early detection of LASV in blood is RT-PCR. Therefore, the European Network for Diagnostics of ‘Imported’ Viral Diseases (ENIVD) performed an external quality assessment (EQA) study for molecular detection of LASV. A proficiency panel of 13 samples containing various concentrations of inactivated LASV strains Josiah, Lib-1580/121, CSF, or AV was prepared. Samples containing the LASV-related lymphocytic choriomeningitis virus (LCMV) and negative sera were included as specificity controls. Twenty-four laboratories from 17 countries (13 European, one African, one Asian, two American countries) participated in the study. Thirteen laboratories (54%) reported correct results, 4 (17%) laboratories reported 1 to 2 false-negative results, and 7 (29%) laboratories reported 3 to 5 false-negative results. This EQA study indicates that most participating laboratories have a good or acceptable performance in molecular detection of LASV. However, several laboratories need to review and improve their diagnostic procedures.     

Is Meat Flavor a Factor in Hunters’ Prey Choice Decisions?

By focusing on the caloric composition of hunted prey species, optimal foraging research has shown that hunters usually make economically rational prey choice decisions. However, research by meat scientists suggests that the gustatory appeal of wildlife meats may vary dramatically. In this study, behavioral research indicates that Mayangna and Miskito hunters in Nicaragua inconsistently pursue multiple prey types in the optimal diet set. We use cognitive methods, including unconstrained pile sorts and cultural consensus analysis, to investigate the hypothesis that these partial preferences are influenced by considerations of meat flavor. Native informants exhibit high agreement on the relative appeal of different meats. Given the absence of other noteworthy differences between spider monkeys (Ateles geoffroyi) and howler monkeys (Alouatta palliata), the unappealing flavor of howler monkeys seems to be a factor in the partial preference for this species.     

Absence of male reproductive skew,along with high frequency of polyandry and conspecific brood parasitism in the lekking Houbara bustard Chlamydotis undulata undulata

Behavioural studies have led to the perception that lekking species experience a high male reproductive skew as a consequence of females’ selective mate choice. In addition, observations suggest that females copulate only once and therefore polyandry seems unlikely as females are supposed to choose the best male available. In order to analyse the mating strategy of the Houbara bustard, an endangered lekking species under reinforcement in eastern Morocco, we used microsatellite data to perform paternity analyses. None of our observations followed common expectations under a lek mating system: we found no male reproductive skew suggesting no apparent selective female mate choice and no apparent male benefit from lekking. In contrast, a high level of polyandry (60 % of the nests) was recorded suggesting that sperm competition may operate. In addition, we present another case of conspecific brood parasitism in a lekking species and this was an unexpected alternative strategy for a species presenting high parental cost and low fecundity. The increasing number of studies contradicting common assumptions on lekking species suggests that alternative breeding strategies such as males pursuing an off‐lek mating strategy, female polyandry and even conspecific brood parasitism might be more widespread in lekking species than previously thought.     

Comparative analysis of virulence genes, genetic diversity, and phylogeny of commensal and enterotoxigenic Escherichia coli isolates from weaned pigs

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (alpha-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.     

Effect of the purinergic receptor P2X7 on Chlamydia infection in cervical epithelial cells and vaginally infected mice

Ligation of the purinergic receptor, P2X7R, with its agonist ATP has been previously shown to inhibit intracellular infection by chlamydiae and mycobacteria in macrophages. The effect of P2X7R on chlamydial infection had never been investigated in the preferred target cells of chlamydiae, cervical epithelial cells, nor in vaginally infected mice. In this study, we show that treatment of epithelial cells with P2X7R agonists inhibits partially Chlamydia infection in epithelial cells. Chelation of ATP with magnesium or pretreatment with a P2X7R antagonist blocks the inhibitory effects of ATP. Similarly to previous results obtained with macrophages, ATP-mediated inhibition of infection in epithelial cells requires activation of host-cell phospholipase D. Vaginal infection was also more efficient in P2X7R-deficient mice, which also displayed a higher level of acute inflammation in the endocervix, oviduct, and mesosalpingeal tissues than in infected wild-type mice. However, secretion of IL-1beta, which requires P2X7R ligation during infection by other pathogens, was decreased mildly and only at short times of infection. Taken together, these results suggest that P2X7R affects Chlamydia infection by directly inhibiting infection in epithelial cells, rather than through the ability of P2X7R to modulate IL-1beta secretion.