Insights into microRNA-mediated interaction and regulation of metabolites in tomato.

microRNAs direct regulation of various metabolic pathways in plants and animals. miRNAs may be useful in developing novel/elite genotypes, with enhanced metabolites and disease resistance. We examined miRNAs in tomato. In tomato, miRNAs in the carotenoid pathway have not been fully elucidated. We examined the potential role of miRNAs in biosynthesis of carotenoids, transcript profiling of miRNAs and their possible targets (genes and transcription factors) at different development stages of tomato using stem-loop PCR and RT-qPCR. We also identified miRNAs targeting key flavonoid genes, such as chalcone isomerase (CHI), and dihydroflavonol-4-reductase (DFR). Distinct expression profiles of miRNAs and their targets were found in fruits of three tomato accessions, suggesting carotenoid regulation by miRNAs at various stages of fruit development. This was also confirmed using HPLC of the carotenoids. The present study may help in understanding possible regulation of carotenoid biosynthesis. The identified miRNAs can be exploited to enhance biosynthesis of different carotenoids in plants.

Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

Is the Management of Rectal Cancer Using a Watch and Wait Approach Feasible, Safe and Effective in a Publicly Funded General Hospital?

AIMS: Although there is emerging evidence to suggest equivalent oncological outcomes using a watch and wait approach compared with primary total mesorectal excision surgery, there is a paucity of evidence about the safety and efficacy of this approach in routine clinical practice. Here we report the long-term outcomes and quality of life from patients managed with watch and wait following a clinical complete response (cCR) to neoadjuvant therapy. MATERIALS AND METHODS: Patients with adenocarcinoma of the rectum with cCR following neoadjuvant therapy managed using watch and wait were retrospectively identified. Demographic data, performance status, pretreatment staging information, oncological and surgical outcomes were obtained from routinely collected clinical data. Quality of life was measured by trained clinicians during telephone interviews. RESULTS: Over a 7-year period, 506 patients were treated for rectal cancer, 276 had neoadjuvant therapy and 72 had a cCR (26.1%). Sixty-three were managed with watch and wait. Thirteen patients had mucosal regrowth. There was no significant difference in the incidence of metastatic disease between the surgical and watch and wait cohorts (P = 0.38). The 13 patients with mucosal regrowth underwent salvage surgery. Eleven of the patients who underwent surgical resection had R0 resections. There was also a statistically and clinically significant improvement in the Functional Assessment of Cancer Therapy - Colorectal (FACT-C) trial outcome index (P = 0.022). CONCLUSION: This study shows that watch and wait is safe and effective outside of tertiary referral centres. It suggests that an opportunistic cCR is durable and when mucosal regrowth occurs it can be salvaged. Finally, we have shown that quality of life is probably improved if a watch and wait approach is adopted.

Allergen Immunotherapy (AIT) in children: a vulnerable population with its own rights and legislation - summary of EMA-initiated multi-stakeholder meeting on Allergen Immunotherapy (AIT) for children, held at Paul-Ehrlich-Institut, Langen, Germany, 16.1.2019.

Concerning development of medicinal products, children belong to a so-called "special population" for which additional legislation applies: Regulation (EC) No 1901/2006 on medicinal products for paediatric use sets up a system of requirements, rewards and incentives to ensure that medicinal products are researched, developed and authorized to meet the therapeutic needs of children. Allergen Immunotherapy (AIT) is believed to contain a strong potential for immunomodulatory effects inducing sustained clinical efficacy after cessation of treatment (disease modifying effect) and thereby may prevent the progression of the atopic march towards asthma manifestation. However, to this day only few data on long-term effects in general exist and even fewer in children. These are predominantly data from open studies, which are strongly influenced in their validity by the known placebo effect of AIT. Furthermore, there are no studies allowing for the conclusion that efficacy in adults are mirrored by a similar efficacy in children and thus, up to now, it is not possible to extrapolate data from adults to children. The Paediatric Committee (PDCO)-European Medicines Agency's (EMA) scientific committee responsible for activities on medicines for children-initiated a Multi-Stakeholder Meeting on AIT for Children held at the Paul-Ehrlich-Institut in Langen, Germany, to provide a platform for discussion and exchange of thoughts to this topic between allergy experts from academia, regulators and AIT-manufacturers. The consented meeting minutes, conclusions and participants are presented.

Gd2Te3: an antiferromagnetic semimetal.

We report high-precision magnetization ([Formula: see text]), magnetic susceptibility ([Formula: see text]), specific heat (C p (T, H)) and 'zero-field' electrical resistivity, [Formula: see text], data taken on Gd2Te3 single crystal over wide ranges of temperature and magnetic field (H), with either [Formula: see text]-axis or [Formula: see text]-plane. [Formula: see text] and [Formula: see text] unambiguously establish that the b-axis is the easy direction of magnetization whereas any direction in the ac-plane is a hard direction. The [Formula: see text]-type anomaly in 'zero-field' specific heat, C p (T, H = 0), and an abrupt drop in [Formula: see text] (characteristic of the paramagnetic (PM) - antiferromagnetic (AFM) phase transition) are observed at the Néel temperature, [Formula: see text] K. [Formula: see text] and C p (T,H) clearly demonstrate that [Formula: see text] shifts to lower temperatures with increasing H irrespective of whether H points in the easy or hard direction. When [Formula: see text], the [Formula: see text] isotherms at temperatures in the range 2.5 K [Formula: see text] [Formula: see text] K reveal the existence of a field-induced spin-flop (SF) transition at fields 4.0 T [Formula: see text] [Formula: see text] [Formula: see text] 4.5 T. The first principles electronic band structure and density of states calculations, based on the density functional theory, correctly predict an AFM ground state (stabilized primarily by the 4f  Gd3+ - 5p  Te2-- 4f  Gd3+ superexchange interactions) and the observed semi-metallic behavior for the Gd2Te3 compound. Moreover, these calculations yield the values [Formula: see text] [Formula: see text] for the ordered magnetic moment per Gd atom at T = 0, [Formula: see text] mJ mol-1 K-2 for the Sommerfeld coefficient for the electronic specific heat contribution and [Formula: see text] K for the Curie-Weiss temperature, respectively. These theoretical estimates conform well with the corresponding experimental values [Formula: see text] [Formula: see text], [Formula: see text] mJ mol-1 K-2 and [Formula: see text] K.

Allergen immunotherapy phase II trials: Challenges in dose finding.

Phase II studies on allergen immunotherapy (AIT) should define the dose with the best balance between efficacy and safety ("optimal dose"). Their key role is based on dose selection for subsequent pivotal studies (phase III, field studies). Since products for AIT differ in composition and unit definitions, phase II trials are mandatory for new products and preparations being developed according to the German Therapy Allergen Ordinance ("Therapie-Allergeneverordnung", TAV) due to current EMA guidelines since 2009. The latter permit various in-vivo models and endpoints for phase II studies, e.g., AIT-induced changes in skin test, nasal, conjunctival or bronchial provocation, or in exposure chamber or field trials. Selection and graduation of the doses, minimization of placebo effects, and sufficient numbers of patients are a challenge. Effort, required time, and costs are important variables for the initiators of phase II trials. Risks are characterized by e.g., a) too small doses without relevant differences compared to placebo, b) missing true dose-response relationships, c) strong placebo effect and consequently small "therapeutic window", d) large heterogeneity and missing distinct differences (compared to placebo), e) too small effects in field studies due to low allergen exposure, f) missing dose-related increase (in case of too high doses). In the view of the Paul-Ehrlich-Institute, the unambiguous phase II trials with TAV products performed until today were not able to confirm the marketed doses for AIT. Regardless of the utilized model, more raw and single data should illustrate the individual outcome of AIT during phase II trials, facilitating an improved and more intuitive interpretation of the data (placebo effects? scattering?). In the medium term, evidence regarding AIT efficacy will considerably increase due to phase II trials as a prerequisite for subsequent phase III field studies. This affects all manufacturers offering AIT products in Germany and Europe.

Identification of novel translocation between short arm of chromosome 4 and long arm of chromosome 6 in an infertile man using Interphase Chromosome Profiling (ICP).

Conventional cytogenetics has always been a favourite to detect chromosomal aberrations. Carriers of chromosomal translocation are often phenotypically normal but are infertile. Couples are often advised to go for karyotyping, but culture failure or improper metaphase spread with poor banding often makes the analysis difficult. We report here a novel translocation between short arm of chromosome 4 and long arm of chromosome 6 in an infertile man using an advanced molecular cytogenetic technique of Interphase Chromosome Profiling (ICP).

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Minor allergen patterns in birch pollen allergen products-A question of pollen?

BACKGROUND: Contrary to the scientific differentiation between major and minor allergens, the regulatory framework controlling allergen products in the EU distinguishes relevant and non-relevant allergens. Given the lack of knowledge on their clinical relevance, minor allergens are usually not controlled by allergen product specifications. Especially, in birch pollen (BP) allergen products, minor allergens are commonly disregarded. OBJECTIVES: To quantify three minor allergens in BP allergen products from different manufacturers and to assess the influence of the utilized BP on minor allergen patterns. METHODS: Apart from common quality parameters such as Bet v 1 content, Bet v 4, Bet v 6 and Bet v 7 were quantified in 70 BP allergen product batches from six manufacturers, using ELISA systems developed in-house. Batch-to-batch variability was checked for agreement with a variability margin of 50%-200% from mean of the given batches for individual allergen content. Subsequently, minor allergen patterns were generated via multidimensional scaling and related to information on the pollen lots used in production of the respective product batches. RESULTS: Like the already established Bet v 4 ELISA, the ELISA systems for quantification of Bet v 6 and Bet v 7 were successfully validated. Differences in minor allergen content between products and batch-to-batch consistency were observed. Correlations between minor and major allergen content were low to moderate. About 20% of batches exceeded the variability margin for at least one minor allergen. Interestingly, these fluctuations could not in all cases be linked to the use of certain BP lots. CONCLUSIONS AND CLINICAL RELEVANCE: The impact of the observed minor allergen variability on safety and efficacy of BP allergen products can currently not be estimated. As the described differences could only in few cases be related to the used pollen lots, it is evident that additional factors influence minor allergens in BP allergen products.

Effects of work-family conflict and job insecurity on psychological distress.

BACKGROUND: Work-family conflict (WFC) and job insecurity are important determinants of workers' mental health. AIMS: To examine the relationship between WFC and psychological distress, and the co-occurring effects of WFC and job insecurity on distress in US working adults. METHODS: This study used cross-sectional data from the 2010 National Health Interview Survey (NHIS) for adults aged 18-64 years. The 2010 NHIS included occupational data from the National Institute for Occupational Safety and Health (NIOSH) sponsored Occupational Health Supplement. Logistic regression models were used to examine the independent and co-occurring effects of WFC and job insecurity on distress. RESULTS: The study group consisted of 12059 participants. In the model fully adjusted for relevant occupational, behavioural, sociodemographic and health covariates, WFC and job insecurity were independently significantly associated with increased odds of psychological distress. Relative to participants reporting WFC only, participants reporting no WFC and no job insecurity had lower odds of moderate and severe distress. Co-occurring WFC and job insecurity was associated with significantly higher odds of both moderate [odds ratio (OR) = 1.55; 95% confidence interval (CI) 1.25-1.9] and severe (OR = 3.57; 95% CI 2.66-4.79) distress. CONCLUSIONS: Rates of WFC and job insecurity were influenced by differing factors in working adults; however, both significantly increased risk of adverse mental health outcomes, particularly when experienced jointly. Future studies should explore the temporal association between co-occurring WFC and job insecurity and psychological distress.

Allergen exposure chambers: harmonizing current concepts and projecting the needs for the future - an EAACI Position Paper.

BACKGROUND: Allergen exposure chambers (AECs) are clinical facilities allowing for controlled exposure of subjects to allergens in an enclosed environment. AECs have contributed towards characterizing the pathophysiology of respiratory allergic diseases and the pharmacological properties of new therapies. In addition, they are complementary to and offer some advantages over traditional multicentre field trials for evaluation of novel therapeutics. To date, AEC studies conducted have been monocentric and have followed protocols unique to each centre. Because there are technical differences among AECs, it may be necessary to define parameters to standardize the AECs so that studies may be extrapolated for driving basic immunological research and for marketing authorization purposes by regulatory authorities. METHODS: For this task force initiative of the European Academy of Allergy and Clinical Immunology (EAACI), experts from academia and regulatory agencies met with chamber operators to list technical, clinical and regulatory unmet needs as well as the prerequisites for clinical validation. RESULTS: The latter covered the validation process, standardization of challenges and outcomes, intra- and interchamber variability and reproducibility, in addition to comparability with field trials and specifics of paediatric trials and regulatory issues. CONCLUSION: This EAACI Position Paper aims to harmonize current concepts in AECs and to project unmet needs with the intent to enhance progress towards use of these facilities in determining safety and efficacy of new therapeutics in the future.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

Epidemiological and survival analysis of triple-negative breast cancer cases in a retrospective multicenter study.

INTRODUCTION: This is a retrospective study with data collected from breast cancer cases from five major Apollo Hospitals across India, as part of a biobanking process. One aspect of our study focused specifically on data from triple-negative breast cancer (TNBC) cases. The aim of this study was to analyze epidemiology, treatment options, and survival of the patients with TNBC. Our goal was to draw conclusions on the preponderance of the disease and also to understand the outcomes using the existing therapy options. MATERIALS AND METHODS: Data were collected after due ethical clearances and were coded with regard to patient identifiers to protect patient privacy. Data were not only from the various departments of the respective hospitals and the treating physicians but also from the follow-up made by hospital staff and social workers. RESULTS: About 20% of all cases of breast cancer comprised TNBC. Although the disease is generally thought to be an early onset disease, there was no major difference in the median age of diagnosis of TNBC compared to other breast cancer cases. More than 85% of the TNBC cases were of early stage disease with <4% of the cases of metastatic cancer. Data on follow-up were somewhat sporadic as a good number of cases were lost to follow-up, but from the available data, recurrence rate was about 11%. Death, when it occurred, was mostly in the early periods of treatment with 35% of the events occurring before 3 years. The overall survival rates beyond 3 years were more than 86%. CONCLUSIONS: Data and sample collection are an ongoing process, so we expect this data set to be enriched with more cases and longer duration of follow-up in a year. Preliminary analysis sheds light on the potential of such a collection both for understanding the epidemiology of the disease and also for conducting future studies with an eye toward improving treatment outcomes.