Identification of biomarkers predictive of metastasis development in early-stage colorectal cancer using network-based regularization.

Colorectal cancer (CRC) is the third most common cancer and the second most deathly worldwide. It is a very heterogeneous disease that can develop via distinct pathways where metastasis is the primary cause of death. Therefore, it is crucial to understand the molecular mechanisms underlying metastasis. RNA-sequencing is an essential tool used for studying the transcriptional landscape. However, the high-dimensionality of gene expression data makes selecting novel metastatic biomarkers problematic. To distinguish early-stage CRC patients at risk of developing metastasis from those that are not, three types of binary classification approaches were used: (1) classification methods (decision trees, linear and radial kernel support vector machines, logistic regression, and random forest) using differentially expressed genes (DEGs) as input features; (2) regularized logistic regression based on the Elastic Net penalty and the proposed iTwiner-a network-based regularizer accounting for gene correlation information; and (3) classification methods based on the genes pre-selected using regularized logistic regression. Classifiers using the DEGs as features showed similar results, with random forest showing the highest accuracy. Using regularized logistic regression on the full dataset yielded no improvement in the methods' accuracy. Further classification using the pre-selected genes found by different penalty factors, instead of the DEGs, significantly improved the accuracy of the binary classifiers. Moreover, the use of network-based correlation information (iTwiner) for gene selection produced the best classification results and the identification of more stable and robust gene sets. Some are known to be tumor suppressor genes (OPCML-IT2), to be related to resistance to cancer therapies (RAC1P3), or to be involved in several cancer processes such as genome stability (XRCC6P2), tumor growth and metastasis (MIR602) and regulation of gene transcription (NME2P2). We show that the classification of CRC patients based on pre-selected features by regularized logistic regression is a valuable alternative to using DEGs, significantly increasing the models' predictive performance. Moreover, the use of correlation-based penalization for biomarker selection stands as a promising strategy for predicting patients' groups based on RNA-seq data.

Impaired anti-HBV vaccine response in non-cirrhotic chronic HCV is not overcome by double dose regimen: randomized control trial.

INTRODUCTION AND OBJECTIVES: Some studies suggest chronic HCV infection diminishes responses to the anti-HBV vaccine. We evaluated the efficacy of double versus standard dose HBV vaccination among HCV patients without cirrhosis. PATIENTS AND METHODS: 141 adults with untreated chronic HCV were randomized to HBV vaccination with double dose (40µg) or standard dose (20µg) at 0, 1 and 6 months; 70 healthy HCV-negative patients given standard dose served as controls. Vaccine response was defined by anti-HBs ≥10 mIU/mL. RESULTS: 128 patients (60 double, 68 standard doses) completed the study. Patients were of median age 52 years, 61% female, 60% fibrosis <2 of 4, and 76% genotype 1 with median 6-log 10 IU/mL HCV RNA. Overall seroprotection rate was 76.7% (95% CI: 65-87) in the 40µg versus 73.5% (95% CI: 63-84) in the 20µg dose HCV-positive groups (p =0.68) and 91.2% (95%CI:84-99) in HCV-negative controls (p =0.011 and 0.003, respectively). In multivariate logistic regression, vaccine dose (double vs. standard dose) was not associated with vaccine response (OR=0.63, p =0.33). Of 32 HCV-infected patients who were non-responders to 3- doses, 25 received the fourth dose of vaccine. The fourth dose seroconversion rate for the 40µg and 20µg groups were 45.5% and 21.4%, respectively. CONCLUSIONS: In HCV-infected patients without cirrhosis, impaired responses to HBV vaccination cannot be overcome by the use of double dose HBV vaccination, but adding a fourth dose of vaccine for non-responders may be an effective strategy. Other adjuvant measures are needed to enhance seroconversion rates in these patients. TRIAL REGISTER: U 1111-1264-2343 (www.ensaiosclinicos.gov.br).

Discovering Loci for Breeding Prospective and Phenology in Wheat Mediterranean Landraces by Environmental and eigenGWAS.

Knowledge of the genetic basis of traits controlling phenology, differentiation patterns, and environmental adaptation is essential to develop new cultivars under climate change conditions. Landrace collections are an appropriate platform to study the hidden variation caused by crop breeding. The use of genome-wide association analysis for phenology, climatic data and differentiation among Mediterranean landraces led to the identification of 651 marker-trait associations that could be grouped in 46 QTL hotspots. A candidate gene analysis using the annotation of the genome sequence of the wheat cultivar 'Chinese Spring' detected 1097 gene models within 33 selected QTL hotspots. From all the gene models, 42 were shown to be differentially expressed (upregulated) under abiotic stress conditions, and 9 were selected based on their levels of expression. Different gene families previously reported for their involvement in different stress responses were found (protein kinases, ras-like GTP binding proteins and ethylene-responsive transcription factors). Finally, the synteny analysis in the QTL hotspots regions among the genomes of wheat and other cereal species identified 23, 21 and 7 ortho-QTLs for Brachypodium, rice and maize, respectively, confirming the importance of these loci.

Strong response after fourth dose of mRNA COVID-19 vaccine in autoimmune rheumatic diseases patients with poor response to inactivated vaccine.

OBJECTIVES: To assess immunogenicity of a heterologous fourth dose of an mRNA (BNT162b2) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in autoimmune rheumatic diseases (ARD) patients with poor/non-response to inactivated vaccine (Sinovac-CoronaVac). METHODS: A total of 164 ARD patients who were coronavirus disease 2019 (COVID-19) poor/non-responders (negative anti-SARS-CoV-2 S1/S2 IgG and/or neutralizing antibodies-NAb) to the third dose of Sinovac-CoronaVac received an additional heterologous dose of mRNA (BNT162b2) 3 months after last dose. IgG and NAb were evaluated before and after the fourth dose. RESULTS: Significant increases were observed after the fourth dose in IgG (66.4 vs 95.1%, P < 0.001), NAb positivity (5.5 vs 83.5%, P < 0.001) and geometric mean titre (29.5 vs 215.8 AU/ml, P < 0.001), and 28 (17.1%) remained poor/non-responders. Patients with negative IgG after a fourth dose were more frequently under rituximab (P = 0.001). Negative NAb was associated with older age (P = 0.015), RA (P = 0.002), SSc (P = 0.026), LEF (P = 0.016) and rituximab use (P = 0.007). In multiple logistic regression analysis, prednisone dose ≥7.5 mg/day (OR = 0.34; P = 0.047), LEF (OR = 0.32, P = 0.036) and rituximab use (OR = 0.19, P = 0.022) were independently associated with negative NAb after the fourth vaccine dose. CONCLUSIONS: This is the largest study to provide evidence of a remarkable humoral response after the fourth dose of heterologous mRNA SARS-CoV-2 vaccination in ARD patients with poor/non-response to the third dose of an inactivated vaccine. We further identified that treatment, particularly rituximab and prednisone, impaired antibody response to this additional dose. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, CoronavRheum #NCT04754698.

Enhancing the resilience of Zostera noltei seagrass meadows against Arenicola spp. bio-invasion: A decision-making approach.

Seagrass meadows provide important and valuable ecosystem services. They are affected by several natural and human-induced stressors, but a combination of natural recovery and management actions have recently inverted the worldwide reduction. The main objectives of this study were to provide science-based knowledge on ecology and restoration, framed on environmental-related policies. By coupling the general guidelines with practical experience, obtained from sequential in situ experiments carried out for several months in a show-case study area, this study provides guidelines useful for restoration practitioners. A decision-making approach is proposed to answer the following questions: 1) What is the best Zostera noltei transplanting method? 2) What is the best technique to reduce the bioturbation activity of Arenicola spp.?, 3) Do bioturbation reduction techniques affect the survival rate of Z. noltei transplants?, and finally, 4) What are the key steps to maximize the success of a Z. noltei transplant and increase the species' resilience? Having a Portuguese coastal lagoon as show-case (Mira Channel, Ria de Aveiro), different transplant and restoration methodologies were tested (i.e. metal frames, nails, bamboo sticks, shoots inserted unanchored into the sediment, and intact units of sediment with seagrasses, named as SODs) to assure low environmental impact on donor meadows, high survival rate of transplanted shoots and the recovery of fragmented or lost meadows. Moreover, to potentially reverse a degraded Arenicola spp. colonized seagrass habitat, different types of natural membranes were tested. Results showed that the best transplanting method is the use of SODs as the self-facilitation process of Z. noltei is enhanced, while being the least invasive for the donor population. The use of a natural membrane can significantly decrease the bioturbation stress caused by Arenicola spp., with jute membrane being the best option, given its cost-handling-benefit trade-offs. Enhancing the success of seagrass restoration requires the implementation of effective measures by environmental restoration practitioners. We defined a three-step process to improve the resilience of Z. noltei. This stepwise approach consists on 1) Characterization of the donor population, 2) Identification of the constraints and implementation of measures to prevent them, and 3) Scale-up the restoration plan. The application of this stepwise approach in intertidal coastal and estuarine systems management will, therefore, facilitate the success of Z. noltei restoration plans.

Evaluation of antimicrobial susceptibility testing of Nocardia spp. isolates by broth microdilution with resazurin and spectrophotometry.

BACKGROUND: Nocardia species are ubiquitous in natural environments and can cause nocardiosis. In the present study, the use of Resazurin salt and Spectrophotometry were proposed as alternative methods to reduce subjectivity in the interpretation of susceptibility results to antimicrobials by the broth microdilution method for Nocardia spp. RESULTS: The susceptibility of Nocardia spp. isolates to Amikacin, Ciprofloxacin, Minocycline and Trimethoprim-Sulfamethoxazole was evaluated by Minimum Inhibitory Concentration (MIC) determinations by the broth microdilution method. To verify cellular growth, the colour-changing dye Resazurin was applied, the Optical Densities were measured on a spectrophotometer, and both were compared to Clinical and Laboratory Standards Institute (CLSI) Gold Standard method (visual MIC determination). Percentages of essential and categorical agreements and interpretative categorical errors were calculated within each method (intra-reading) and between them (inter-reading). The Gold Standard visual reading demonstrated 100% of essential and categorical intra-reading agreements for Amikacin, and there was no error when compared with the alternative methods. For Ciprofloxacin, the comparison between the Gold Standard and the Spectrophotometric reading showed 91.5% of essential agreement. In the categorical intra-reading analysis for Minocycline, there were 88.1 and 91.7% in the Gold Standard and in the Spectrophotometric readings, respectively, and 86.4% of concordance between them. High rates of categorical agreement were also observed on the Trimethoprim-Sulfamethoxazole analyses, with 93.7% for the Gold Standard, 84.9% for the Resazurin readings, and 80.5% between them. CONCLUSIONS: The alternative methods with Resazurin and Spectrophotometric readings showed high agreement rates with the Gold Standard.

Tracking intratumoral heterogeneity in glioblastoma via regularized classification of single-cell RNA-Seq data.

BACKGROUND: Understanding cellular and molecular heterogeneity in glioblastoma (GBM), the most common and aggressive primary brain malignancy, is a crucial step towards the development of effective therapies. Besides the inter-patient variability, the presence of multiple cell populations within tumors calls for the need to develop modeling strategies able to extract the molecular signatures driving tumor evolution and treatment failure. With the advances in single-cell RNA Sequencing (scRNA-Seq), tumors can now be dissected at the cell level, unveiling information from their life history to their clinical implications. RESULTS: We propose a classification setting based on GBM scRNA-Seq data, through sparse logistic regression, where different cell populations (neoplastic and normal cells) are taken as classes. The goal is to identify gene features discriminating between the classes, but also those shared by different neoplastic clones. The latter will be approached via the network-based twiner regularizer to identify gene signatures shared by neoplastic cells from the tumor core and infiltrating neoplastic cells originated from the tumor periphery, as putative disease biomarkers to target multiple neoplastic clones. Our analysis is supported by the literature through the identification of several known molecular players in GBM. Moreover, the relevance of the selected genes was confirmed by their significance in the survival outcomes in bulk GBM RNA-Seq data, as well as their association with several Gene Ontology (GO) biological process terms. CONCLUSIONS: We presented a methodology intended to identify genes discriminating between GBM clones, but also those playing a similar role in different GBM neoplastic clones (including migrating cells), therefore potential targets for therapy research. Our results contribute to a deeper understanding on the genetic features behind GBM, by disclosing novel therapeutic directions accounting for GBM heterogeneity.

OPTICAL COHERENCE TOMOGRAPHY LEAKAGE IN NEOVASCULAR AGE-RELATED MACULAR DEGENERATION: Identification of Choroidal Neovascularization Activity by Location and Quantification of Abnormal Fluid Under Anti-Vascular Endothelial Growth Factor Therapy.

PURPOSE: To test optical coherence tomography leakage in the identification and quantification of choroidal neovascularization-related fluid, its change after anti-vascular endothelial growth factor therapy in neovascular age-related macular degeneration eyes and its relation to functional outcome. METHODS: Prospective analysis of a cohort of neovascular age-related macular degeneration cases treated with 2.0-mg intravitreal aflibercept. Eyes included were analyzed before, 1-week, and 1-month after one injection. Best-corrected visual acuity was assessed using Early Treatment Diabetic Retinopathy Study method. Optical coherence tomography leakage maps depicting low optical reflectivity (LOR) sites were acquired with OCT Cirrus AngioPlex (Zeiss, Dublin, CA). The LOR area ratio was correlated to retinal thickness and best-corrected visual acuity. Optical coherence tomography angiography was simultaneously performed. RESULTS: Twenty-two eyes of 18 patients with neovascular age-related macular degeneration were included. The LOR ratio of the full retina scan and retinal pigment epithelium-Bruch layer decreased from baseline to Month 1 (P < 0.05). Changes in retinal thickness and LOR ratio were positively correlated (P < 0.05). Best-corrected visual acuity change correlated with the outer segment layer LOR change (rho = -0.53, P = 0.014), and LOR was inferior in better responders (P = 0.021). Optical coherence tomography leakage identified eyes with recurrent fluid in the external layers. CONCLUSION: Optical coherence tomography leakage identified and quantified the fluid related to choroidal neovascularization activity. Low optical reflectivity change in the outer segment layer correlates with functional outcome and increasing LOR in the external layers may be a marker of early recurrence. Combining optical coherence tomography angiography and optical coherence tomography leakage allows both for choroidal neovascularization morphology and activity analysis.

Twiner: correlation-based regularization for identifying common cancer gene signatures.

BACKGROUND: Breast and prostate cancers are typical examples of hormone-dependent cancers, showing remarkable similarities at the hormone-related signaling pathways level, and exhibiting a high tropism to bone. While the identification of genes playing a specific role in each cancer type brings invaluable insights for gene therapy research by targeting disease-specific cell functions not accounted so far, identifying a common gene signature to breast and prostate cancers could unravel new targets to tackle shared hormone-dependent disease features, like bone relapse. This would potentially allow the development of new targeted therapies directed to genes regulating both cancer types, with a consequent positive impact in cancer management and health economics. RESULTS: We address the challenge of extracting gene signatures from transcriptomic data of prostate adenocarcinoma (PRAD) and breast invasive carcinoma (BRCA) samples, particularly estrogen positive (ER+), and androgen positive (AR+) triple-negative breast cancer (TNBC), using sparse logistic regression. The introduction of gene network information based on the distances between BRCA and PRAD correlation matrices is investigated, through the proposed twin networks recovery (twiner) penalty, as a strategy to ensure similarly correlated gene features in two diseases to be less penalized during the feature selection procedure. CONCLUSIONS: Our analysis led to the identification of genes that show a similar correlation pattern in BRCA and PRAD transcriptomic data, and are selected as key players in the classification of breast and prostate samples into ER+ BRCA/AR+ TNBC/PRAD tumor and normal tissues, and also associated with survival time distributions. The results obtained are supported by the literature and are expected to unveil the similarities between the diseases, disclose common disease biomarkers, and help in the definition of new strategies for more effective therapies.

Ensemble outlier detection and gene selection in triple-negative breast cancer data.

BACKGROUND: Learning accurate models from 'omics data is bringing many challenges due to their inherent high-dimensionality, e.g. the number of gene expression variables, and comparatively lower sample sizes, which leads to ill-posed inverse problems. Furthermore, the presence of outliers, either experimental errors or interesting abnormal clinical cases, may severely hamper a correct classification of patients and the identification of reliable biomarkers for a particular disease. We propose to address this problem through an ensemble classification setting based on distinct feature selection and modeling strategies, including logistic regression with elastic net regularization, Sparse Partial Least Squares - Discriminant Analysis (SPLS-DA) and Sparse Generalized PLS (SGPLS), coupled with an evaluation of the individuals' outlierness based on the Cook's distance. The consensus is achieved with the Rank Product statistics corrected for multiple testing, which gives a final list of sorted observations by their outlierness level. RESULTS: We applied this strategy for the classification of Triple-Negative Breast Cancer (TNBC) RNA-Seq and clinical data from the Cancer Genome Atlas (TCGA). The detected 24 outliers were identified as putative mislabeled samples, corresponding to individuals with discrepant clinical labels for the HER2 receptor, but also individuals with abnormal expression values of ER, PR and HER2, contradictory with the corresponding clinical labels, which may invalidate the initial TNBC label. Moreover, the model consensus approach leads to the selection of a set of genes that may be linked to the disease. These results are robust to a resampling approach, either by selecting a subset of patients or a subset of genes, with a significant overlap of the outlier patients identified. CONCLUSIONS: The proposed ensemble outlier detection approach constitutes a robust procedure to identify abnormal cases and consensus covariates, which may improve biomarker selection for precision medicine applications. The method can also be easily extended to other regression models and datasets.

Correction to: Genetic analysis of multi-environmental spring wheat trials identifies genomic regions for locus-specific trade-offs for grain weight and grain number.

Unfortunately, the Fig. 1 of this original article was incorrectly published. The corrected Fig. 1 is given below.

Genetic analysis of multi-environmental spring wheat trials identifies genomic regions for locus-specific trade-offs for grain weight and grain number.

KEY MESSAGE: GWAS on multi-environment data identified genomic regions associated with trade-offs for grain weight and grain number. Grain yield (GY) can be dissected into its components thousand grain weight (TGW) and grain number (GN), but little has been achieved in assessing the trade-off between them in spring wheat. In the present study, the Wheat Association Mapping Initiative (WAMI) panel of 287 elite spring bread wheat lines was phenotyped for GY, GN, and TGW in ten environments across different wheat growing regions in Mexico, South Asia, and North Africa. The panel genotyped with the 90 K Illumina Infinitum SNP array resulted in 26,814 SNPs for genome-wide association study (GWAS). Statistical analysis of the multi-environmental data for GY, GN, and TGW observed repeatability estimates of 0.76, 0.62, and 0.95, respectively. GWAS on BLUPs of combined environment analysis identified 38 loci associated with the traits. Among them four loci-6A (85 cM), 5A (98 cM), 3B (99 cM), and 2B (96 cM)-were associated with multiple traits. The study identified two loci that showed positive association between GY and TGW, with allelic substitution effects of 4% (GY) and 1.7% (TGW) for 6A locus and 0.2% (GY) and 7.2% (TGW) for 2B locus. The locus in chromosome 6A (79-85 cM) harbored a gene TaGW2-6A. We also identified that a combination of markers associated with GY, TGW, and GN together explained higher variation for GY (32%), than the markers associated with GY alone (27%). The marker-trait associations from the present study can be used for marker-assisted selection (MAS) and to discover the underlying genes for these traits in spring wheat.

Influence of neutrophil-lymphocyte ratio in prognosis of glioblastoma multiforme.

Neutrophil-lymphocyte ratio (NLR) is a hematological marker of systemic inflammation and several studies demonstrate an association between a higher NLR and a worse prognosis in many malignancies. However, literature analyzing its prognostic value in glioblastoma multiforme (GBM) is still scarce. We intended to analyze the correlation of NLR with overall survival and progression-free survival in patients with GBM performing a retrospective review of the patients with diagnosis of GBM submitted to a resection surgery in the department of neurosurgery of a tertiary care hospital, between January/2005 and January/2013. 140 patients were included. Mean age at surgery was 62.9 ± 10.0 years and mean age at death was 64.4 ± 9.8 years. Mean overall survival was 19.4 ± 14.3 months and mean progression-free survival was 9.4 ± 8.7 months. There was no correlation of NLR, platelets-lymphocyte ratio (PLR) or absolute counts of neutrophils, lymphocytes and platelets with overall survival in multivariate analysis. However, a preoperative NLR ≤ 5 correlated with a shorter progression-free survival [HR 1.56 (SD 95% 1.04-2.34); p = 0.032]. We performed a subgroup analysis of patients who completed Stupp protocol. In this subgroup of 117 patients, a preoperative NLR > 7 correlated with a shorter overall survival [HR 1.65 (SD 95% 1.07-2.53); p = 0.023]. The results from our total cohort didn't confirm the correlation between a higher NRL and worse survival in GBM. However, in the subgroup analysis of patients who completed Stupp protocol, a higher NLR was an independent prognostic factor to a shorter overall survival, similar to existent literature data about GBM.

Multimodal Evaluation of the Fellow Eye of Patients with Retinal Angiomatous Proliferation.

INTRODUCTION: We conducted a multimodal, cross-sectional evaluation. METHODS: Eyes were divided into 4 study groups: controls, early/intermediate age-related macular degeneration (AMD), fellow eyes of retinal angiomatous proliferation (RAP), and RAP eyes. Patients were evaluated with spectral-domain optical coherence tomography (OCT), enhanced depth imaging-OCT, and OCT angiography (OCTA). OCTA images were processed to generate maps of the vessel density and perfusion density of the superficial and deep retinal layers (SRL and DRL) and the choriocapillaris level (CL). The thickness of the outer nuclear layer and choroid was manually assessed. RESULTS: We included 135 eyes of 100 patients (51 controls, 30 AMD, 42 RAP, and 12 fellow eyes). The fellow eyes showed a significantly lower vascular perfusion of the SRL, DRL, and CL (p < 0.02) than the early/intermediate AMD and control eyes did. Similarly, RAP eyes presented a lower vascular perfusion of the DRL and CL (p < 0.05). Besides, structural analyses of the fellow eyes and RAP eyes revealed a significantly higher prevalence of macular pigmentary changes, atrophy of the retinal pigment epithelium, hyperreflective "clumps" above flat drusen, amongst others, than early/intermediate AMD and control eyes (p < 0.05). CONCLUSION: We present the first report on the OCTA analysis of the fellow eye of patients with RAP. The reduced perfusion density and vessel density observed contributes, in association with clearly defined structural changes, to a wider characterization of RAP as a distinctive phenotype.

Pre-travel malaria chemoprophylaxis counselling in a public travel medicine clinic in São Paulo, Brazil.

BACKGROUND: Malaria is one of the most prevalent parasitic diseases in the world and represents a threat to travellers visiting endemic areas. Chemoprophylaxis is the prevention measure used in travel medicine, avoiding clinical manifestations and protecting against the development of severe disease and death. METHODS: Retrospective and descriptive analysis of malaria prevention data in travellers was recorded from a travel medicine clinic in São Paulo, Brazil, between January 2006 and December 2010. All the medical records of travellers, who had travelled to areas with risk of disease transmission, including Brazil, were analysed. Demographic characteristics of travellers, travel details and recommendations for preventing malaria were also seen. RESULTS: During the study period, 2836 pre-travel consultations were carried out on 2744 individuals (92 were consulted twice). The most common reasons for travelling were tourism and work. The most common destinations were Africa (24.5%), Europe (21.2%), Asia (16.6%) and locations within Brazil (14.9%). In general prophylaxis against malaria was recommended in 10.3% of all the consultations. African destinations vs Asian, Brazilian and other destinations and length of stay ≤30 days were independently associated with the higher odds of chemoprophylaxis recommendation after the logistic regression. CONCLUSION: The prophylaxis against malaria was recommended in 10.3% of the consultations. The authors believe that a coherent measure of malaria prevention in Brazil and for international travellers would be to recommend for all parts of the North Brazil, avoidance of mosquito bites and immediate consultation of a physician in case of fever during or after the journey is recommended.

High-throughput FTIR-based bioprocess analysis of recombinant cyprosin production.

To increase the knowledge of the recombinant cyprosin production process in Saccharomyces cerevisiae cultures, it is relevant to implement efficient bioprocess monitoring techniques. The present work focuses on the implementation of a mid-infrared (MIR) spectroscopy-based tool for monitoring the recombinant culture in a rapid, economic, and high-throughput (using a microplate system) mode. Multivariate data analysis on the MIR spectra of culture samples was conducted. Principal component analysis (PCA) enabled capturing the general metabolic status of the yeast cells, as replicated samples appear grouped together in the score plot and groups of culture samples according to the main growth phase can be clearly distinguished. The PCA-loading vectors also revealed spectral regions, and the corresponding chemical functional groups and biomolecules that mostly contributed for the cell biomolecular fingerprint associated with the culture growth phase. These data were corroborated by the analysis of the samples' second derivative spectra. Partial least square (PLS) regression models built based on the MIR spectra showed high predictive ability for estimating the bioprocess critical variables: biomass (R 2 = 0.99, RMSEP 2.8%); cyprosin activity (R 2 = 0.98, RMSEP 3.9%); glucose (R 2 = 0.93, RMSECV 7.2%); galactose (R 2 = 0.97, RMSEP 4.6%); ethanol (R 2 = 0.97, RMSEP 5.3%); and acetate (R 2 = 0.95, RMSEP 7.0%). In conclusion, high-throughput MIR spectroscopy and multivariate data analysis were effective in identifying the main growth phases and specific cyprosin production phases along the yeast culture as well as in quantifying the critical variables of the process. This knowledge will promote future process optimization and control the recombinant cyprosin bioprocess according to Quality by Design framework.

Modelling and genetic dissection of staygreen under heat stress.

KEY MESSAGE: Staygreen traits are associated with heat tolerance in bread wheat. QTL for staygreen and related traits were identified across the genome co-located with agronomic and physiological traits associated to plant performance under heat stress. Plant chlorophyll retention-staygreen-is considered a valuable trait under heat stress. Five experiments with the Seri/Babax wheat mapping population were sown in Mexico under hot-irrigated environments. Normalized difference vegetation index (NDVI) during plant growth was measured regularly and modelled to capture the dynamics of plant greenness decay, including staygreen (Stg) at physiological maturity which was estimated by regression of NDVI during grainfilling. The rate of senescence, the percentage of plant greenness decay, and the area under the curve were also estimated based on NDVI measurements. While Stg and the best fitted curve were highly environment dependent, both traits showed strong (positive for Stg) correlations with yield, grainfilling rates, and extended grainfilling periods, while associations with kernel number and kernel weight were weak. Stg expression was largely dependent on rate of senescence which was related to the pattern of the greenness decay curve and the initial NDVI. QTL analyses revealed a total of 44 loci across environments linked to Stg and related traits, distributed across the genome, with the strongest and most repeatable effects detected on chromosomes 1B, 2A, 2B, 4A, 4B and 7D. Of these, some were common with regions controlling phenology but independent regions were also identified. The co-location of QTL for Stg and performance traits in this study confirms that the staygreen phenotype is a useful trait for productivity enhancement in hot-irrigated environments.

Exploiting genetic diversity from landraces in wheat breeding for adaptation to climate change.

Climate change has generated unpredictability in the timing and amount of rain, as well as extreme heat and cold spells that have affected grain yields worldwide and threaten food security. Sources of specific adaptation related to drought and heat, as well as associated breeding of genetic traits, will contribute to maintaining grain yields in dry and warm years. Increased crop photosynthesis and biomass have been achieved particularly through disease resistance and healthy leaves. Similarly, sources of drought and heat adaptation through extended photosynthesis and increased biomass would also greatly benefit crop improvement. Wheat landraces have been cultivated for thousands of years under the most extreme environmental conditions. They have also been cultivated in lower input farming systems for which adaptation traits, particularly those that increase the duration of photosynthesis, have been conserved. Landraces are a valuable source of genetic diversity and specific adaptation to local environmental conditions according to their place of origin. Evidence supports the hypothesis that landraces can provide sources of increased biomass and thousand kernel weight, both important traits for adaptation to tolerate drought and heat. Evaluation of wheat landraces stored in gene banks with highly beneficial untapped diversity and sources of stress adaptation, once characterized, should also be used for wheat improvement. Unified development of databases and promotion of data sharing among physiologists, pathologists, wheat quality scientists, national programmes, and breeders will greatly benefit wheat improvement for adaptation to climate change worldwide.

Genome-wide association study for grain yield and related traits in an elite spring wheat population grown in temperate irrigated environments.

KEY MESSAGE: Through genome-wide association study, loci for grain yield and yield components were identified in chromosomes 5A and 6A in spring wheat (Triticum aestivum). Genome-wide association study (GWAS) was conducted for grain yield (YLD) and yield components on a wheat association mapping initiative (WAMI) population of 287 elite, spring wheat lines grown under temperate irrigated high-yield potential condition in Ciudad Obregón, Mexico, during four crop cycles (from 2009-2010 to 2012-2013). The population was genotyped with high-density Illumina iSelect 90K single nucleotide polymorphisms (SNPs) assay. An analysis of traits across subpopulations indicated that lines with 1B/1R translocation had higher YLD, grain weight, and taller plants than lines without the translocation. GWAS using 18,704 SNPs identified 31 loci that explained 5-14 % of the variation in individual traits. We identified SNPs in chromosome 5A and 6A that were significantly associated with yield and yield components. Four loci were detected for YLD in chromosomes 3B, 5A, 5B, and 6A and the locus in 5A explained 5 % of the variation for grain number/m(2). The locus for YLD in chromosome 6A also explained 6 % of the variation in grain weight. Loci significantly associated with maturity were identified in chromosomes 2B, 3B, 4B, 4D, and 6A and for plant height in 1A and 6A. Loci were also detected for canopy temperature at grain filling (2D, 4D, 6A), chlorophyll index at grain filling (3B and 6A), biomass (3D and 6A) and harvest index (1D, 1B, and 3B) that explained 5-10 % variation. These markers will be further validated.

A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses.

Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R (2) ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R (2) = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.