Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

Zeugodacus cucumis and Bactrocera jarvisi are pests of fruit and vegetable crops and cause damage to horticulture industries. Immature stages of these two fruit fly species have been intercepted in New Zealand a number of times. Identification to species was not possible using morphological characters; thus, it is important to develop an assay for their species‐level identification. Here, the real‐time PCR assays for rapid identification of Z. cucumis and B. jarvisi were developed and validated. The PCR protocols demonstrated their specificity by amplifying the two target species successfully, with no cross‐reactions observed in the tested tephritid species. The in silico test of the primer and probe binding sites of the two assays also demonstrated the assays’ specificity by no mismatches present in the binding regions of the target species, but 1–4 mismatches in the binding regions of the non‐target fruit fly species. The thresholds of detection for the two assays are as low as 10 copies/µl of the target DNA, indicating that the assays have a very high sensitivity. The application of the real‐time PCR assays has greatly assisted in routine pest identifications at the New Zealand border and surveillance programme. Therefore, the assays have the potential to be used by diagnostic agencies and research organizations worldwide.     

Analysis of PTEN Complex Assembly and Identification of Heterogeneous Nuclear Ribonucleoprotein C as a Component of the PTEN-associated Complex

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is well characterized for its role in antagonizing the phosphoinositide 3-kinase pathway. Previous studies using size-exclusion chromatography demonstrated PTEN recruitment into high molecular mass complexes and hypothesized that PTEN phosphorylation status and PDZ binding domain may be required for such complex formation. In this study, we set out to test the structural requirements for PTEN complex assembly and identify the component(s) of the PTEN complex(es). Our results demonstrated that the PTEN catalytic function and PDZ binding domain are not absolutely required for its complex formation. On the other hand, PTEN phosphorylation status has a significant impact on its complex assembly. Our results further demonstrate enrichment of the PTEN complex in nuclear lysates, suggesting a mechanism through which PTEN phosphorylation may regulate its complex assembly. These results prompted further characterization of other protein components within the PTEN complex(es). Using size-exclusion chromatography and two-dimensional difference gel electrophoresis followed by mass spectrometry analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a novel protein recruited to higher molecular mass fractions in the presence of PTEN. Further analysis indicates that endogenous hnRNP C and PTEN interact and co-localize within the nucleus, suggesting a potential role for PTEN, alongside hnRNP C, in RNA regulation.Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)⁴ was cloned in 1997 (1–3) and has been well characterized for its tumor-suppressive role by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol 4,5-bisphosphate and antagonizing the phosphoinositide 3-kinase pathway (4–7). PTEN also regulates cell migration, cell cycle progression, DNA damage response, and chromosome stability independently of its lipid phosphatase activity through its potential protein phosphatase activity and/or protein-protein interaction (8–11) (for recent reviews, see 12–14).PTEN is composed of an N-terminal catalytic domain and a C-terminal regulatory domain. The catalytic domain contains a conserved signature motif (HCXXGXXR) found in dual-specific protein phosphatases, and mutations within this catalytic domain, including the C124S mutation, are known to abrogate PTEN catalytic activity (4). The C terminus of PTEN contains a PDZ (PDS-95/Disc-large/Zo-1) binding domain, which interacts with PDZ-containing proteins such as MAGI-1b, MAGI-2, MAGI-3, hDLG, hMAST and NHERF (15–19). In addition to the PDZ binding domain, several key serine and threonine phosphorylation sites (Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵) at the PTEN C terminus are reported to play an important role in regulating its stability, localization, and activity (20–26).Recent studies suggest that PTEN may function within higher molecular mass complexes. Through size-exclusion chromatography, Vazquez et al. (27) demonstrated that PTEN can be separated into two populations: a monomeric hyperphosphorylated subpopulation and a higher molecular mass hypophosphorylated subpopulation. It was hypothesized that PTEN in its dephosphorylated form can interact with PDZ-containing proteins such as hDLG and be recruited into a higher molecular mass complex. Although the components within PTEN complex(es) have not been systematically studied and purified, MAGI-2, hDLG (27), NHERF2, PDGFR (19), NEP (28), and MVP (29) have been identified as potential components of the PTEN complex using the same size-exclusion chromatography methodology.In this paper, we aim to (i) investigate the essential elements of PTEN required for its complex formation and (ii) dissect the components of the PTEN-associated complex(es). Our results indicate that PTEN catalytic activity or its PDZ binding domain is not absolutely required for complex assembly. PTEN phosphorylation status on amino acids Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵, on the other hand, has a significant role in complex formation. In addition, we demonstrate that the PTEN complex is enriched in nuclear lysates, which suggests a mechanism through which phosphorylation can regulate complex assembly. Using two-dimensional difference gel electrophoresis (DIGE) analysis and comparing proteins present in higher molecular mass fractions in the presence and absence of PTEN followed by mass spectrometry analysis, we have identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a potential component within the PTEN complex. PTEN and hnRNP C are shown here to interact and co-localize in the nucleus. We hypothesize that the PTEN and hnRNP C complex may play a role in RNA regulation.     

The application of genetics and nutritional genomics in practice: an international survey of knowledge, involvement and confidence among dietitians in the US, Australia and the UK

As a result of expanding scientific understanding of the interplay between genetics and dietary risk factors, those involved in nutritional management need to understand genetics and nutritional genomics in order to inform management of individuals and groups. The aim of this study was to measure and determine factors affecting dietitians’ knowledge, involvement and confidence in genetics and nutritional genomics across the US, Australia and the UK. A cross-sectional study was undertaken using an online questionnaire that measured knowledge and current involvement and confidence in genetics and nutritional genomics. The questionnaire was distributed to dietitians in the US, Australia and the UK using email lists from the relevant professional associations. Data were collected from 1,844 dietitians who had practiced in the previous 6 months. The main outcomes were knowledge of genetics and nutritional genomics and involvement and confidence in undertaking clinical and educational activities related to genetics and nutritional genomics. Mean scores for knowledge, involvement and confidence were calculated. Analysis of variance and χ ² analysis were used to compare scores and frequencies. Multivariate linear regression was used to determine predictors of high scores. The results demonstrated significant differences in involvement (p < 0.001) and confidence (p < 0.001) but not knowledge scores (p = 0.119) between countries. Overall, dietitians reported low levels of knowledge (mean knowledge score 56.3 %), involvement (mean number of activities undertaken 20.0–22.7 %) and confidence (mean confidence score 25.8–29.7 %). Significant relationships between confidence, involvement and knowledge were observed. Variables relating to education, experience, sector of employment and attitudes were also significantly associated with knowledge, involvement and confidence. Dietitians’ knowledge, involvement and confidence relating to genetics and nutritional genomics remain low and further investigation into factors contributing to this is required.     

Direct discovery and validation of a peptide/MHC epitope expressed in primary human breast cancer cells using a TCRm monoclonal antibody with profound antitumor properties

The identification and validation of new cancer-specific T cell epitopes continues to be a major area of research interest. Nevertheless, challenges remain to develop strategies that can easily discover and validate epitopes expressed in primary cancer cells. Regarded as targets for T cells, peptides presented in the context of the major histocompatibility complex (MHC) are recognized by monoclonal antibodies (mAbs). These mAbs are of special importance as they lend themselves to the detection of epitopes expressed in primary tumor cells. Here, we use an approach that has been successfully utilized in two different infectious disease applications (WNV and influenza). A direct peptide-epitope discovery strategy involving mass spectrometric analysis led to the identification of peptide YLLPAIVHI in the context of MHC A*02 allele (YLL/A2) from human breast carcinoma cell lines. We then generated and characterized an anti-YLL/A2 mAb designated as RL6A TCRm. Subsequently, the TCRm mAb was used to directly validate YLL/A2 epitope expression in human breast cancer tissue, but not in normal control breast tissue. Moreover, mice implanted with human breast cancer cells grew tumors, yet when treated with RL6A TCRm showed a marked reduction in tumor size. These data demonstrate for the first time a coordinated direct discovery and validation strategy that identified a peptide/MHC complex on primary tumor cells for antibody targeting and provide a novel approach to cancer immunotherapy.     

DNA repair defects channel interstrand DNA cross-links into alternate recombinational and error-prone repair pathways

The repair of psoralen interstrand cross-links in the yeast Saccharomyces cerevisiae involves the DNA repair groups nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (PRR). In repair-proficient yeast cells cross-links induce double-strand breaks, in an NER-dependent process; the double-strand breaks are then repaired by HR. An alternate error-prone repair pathway generates mutations at cross-link sites. We have characterized the repair of plasmid molecules carrying a single psoralen cross-link, psoralen monoadduct, or double-strand break in yeast cells with deficiencies in NER, HR, or PRR genes, measuring the repair efficiencies and the levels of gene conversions, crossing over, and mutations. Strains with deficiencies in the NER genes RAD1, RAD3, RAD4, and RAD10 had low levels of cross-link-induced recombination but higher mutation frequencies than repair-proficient cells. Deletion of the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57 also decreased induced recombination and increased mutation frequencies above those of NER-deficient yeast. Strains lacking the PRR genes RAD5, RAD6, and RAD18 did not have any cross-link-induced mutations but showed increased levels of recombination; rad5 and rad6 cells also had altered patterns of cross-link-induced gene conversion in comparison with repair-proficient yeast. Our observations suggest that psoralen cross-links can be repaired by three pathways: an error-free recombinational pathway requiring NER and HR and two PRR-dependent error-prone pathways, one NER-dependent and one NER-independent.     

Fetal heart rate variability and brain stem injury after asphyxia in preterm fetal sheep

This study was undertaken to determine the mechanisms mediating changes in fetal heart rate variability (FHRV) during and after exposure to asphyxia in the premature fetus. Preterm fetal sheep at 0.6 of gestation (91 +/- 1 days, term is 147 days) were exposed to either sham occlusion (n = 10) or to complete umbilical cord occlusion for either 20 (n = 7) or 30 min (n = 10). Cord occlusion led to a transient increase in FHRV with abrupt body movements that resolved after 5 min. In the 30 min group there was a marked increase in FHRV in the final 10 min of occlusion related to abnormal atrial activity. After reperfusion, FHRV in both study groups was initially suppressed and progressively increased to baseline levels over the first 4 h of recovery. In the 20 min group this improvement was associated with return of normal EEG activity and movements. In contrast, in the 30 min group the EEG was abnormal with epileptiform activity superimposed on a suppressed background, which was associated with abnormal fetal movements. As the epileptiform activity resolved, FHRV fell and became suppressed for the remainder of the study. Histological assessment after 72 h demonstrated severe brain stem injury in the 30 min group but not in the 20 min group. In conclusion, during early recovery from asphyxia, epileptiform activity and associated abnormal fetal movements related to evolving neural injury can cause a confounding transient increase in FHRV, which mimics the normal pattern of recovery. However, chronic suppression of FHRV was a strong predictor of severe brain stem injury.