The link between obesity and the gut microbiota and immune system in early-life.

In early-life, the gut microbiota is highly modifiable, being modulated by external factors such as maternal microbiota, mode of delivery, and feeding strategies. The composition of the child's gut microbiota will deeply impact the development and maturation of its immune system, with consequences for future health. As one of the main sources of microorganisms to the child, the mother represents a crucial factor in the establishment of early-life microbiota, impacting the infant's wellbeing. Recent studies have proposed that dysbiotic maternal gut microbiota could be transmitted to the offspring, influencing the development of its immunity, and leading to the development of diseases such as obesity. This paper aims to review recent findings in gut microbiota and immune system interaction in early-life, highlighting the benefits of a balanced gut microbiota in the regulation of the immune system.

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library.

Xylanases are key biocatalysts in the degradation of the ß-1,4-glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-ß-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. KEY POINTS: • A GH10 endo-1,4-ß-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB's in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis.

Presence of pathogen DNA in milk harvested from quarters is associated to changes in cows' milk yield and composition.

BACKGROUND: Intramammary infection is the result of invasion and multiplication of microorganisms in the mammary gland and commonly leads to mastitis in dairy animals. Although much has been done to improve cows' udder health, mastitis remains a significant and costly health issue for dairy farmers, especially if subclinical. In this study, quarter milk samples from clinically healthy cows were harvested to detect pathogens via quantitative PCR (qPCR) and evaluate changes in individual milk traits according to the number of quarters infected and the type of microorganism(s). A commercial qPCR kit was used for detection of Mycoplasma bovis, Mycoplasma spp., Staphylococcus aureus, coagulase-negative staphylococci (CNS), Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Prototheca spp., Escherichia coli, Klebsiella spp., Enterococcus spp. and Lactococcus lactis ssp. lactis. Quarter and pooled milk information of 383 Holstein, 132 Simmental, 129 Rendena, and 112 Jersey cows in 9 Italian single-breed herds was available. RESULTS: Among the cows with pathogen(s) present in at least 1 quarter, CNS was the most commonly detected DNA, followed by Streptococcus uberis, Mycoplasma bovis, and Streptococcus agalactiae. Cows negative to qPCR were 206 and had the lowest milk somatic cell count. Viceversa, cows with DNA isolated in ≥ 3 quarters were those with the highest somatic cell count. Moreover, when major pathogens were isolated in ≥ 3 quarters, milk had the lowest casein index and lactose content. In animals with pathogen(s) DNA isolated, the extent with whom milk yield and major solids were impaired did not significantly differ between major and minor pathogens. CONCLUSIONS: The effect of the number of affected quarters on the pool milk quality traits was investigated in clinically healthy cows using a commercial kit. Results remark the important negative effect of subclinical udder inflammations on milk yield and quality, but more efforts should be made to investigate the presence of untargeted microorganisms, as they may be potentially dangerous for cows. For a smarter use of antimicrobials, analysis of milk via qPCR is advisable - especially in cows at dry off - to identify quarters at high risk of inflammation and thus apply a targeted/tailored treatment.

The ability of a handheld near-infrared spectrometer to do a rapid quality assessment of bovine colostrum, including the immunoglobulin G concentration.

Portable infrared-based instruments have made important contributions in different research fields. Within the dairy supply chain, for example, most of portable devices are based on near-infrared spectroscopy (NIRS) and are nowadays an important support for farmers and operators of the dairy sector, allowing fast and real-time decision-making, particularly for feed and milk quality evaluation and animal health and welfare monitoring. The affordability, portability, and ease of use of these instruments have been pivotal factors for their implementation on farm. In fact, pocket-sized devices enable nonexpert users to perform quick, low-cost, and nondestructive analysis on various matrixes without complex preparation. Because bovine colostrum (BC) quality is mostly given by the IgG level, evaluating the ability of portable NIRS tools to measure antibody concentration is advisable. In this study we used the wireless device SCiO manufactured by Consumer Physics Inc. (Tel Aviv, Israel) to collect BC spectra and then attempt to predict IgG concentration and gross and fine composition in individual samples collected immediately after calving (<6 h) in primiparous and pluriparous Holstein cows on 9 Italian farms. Chemometric analyses revealed that SCiO has promising predictive performance for colostral IgG concentration, total Ig concentration, fat, and AA. The coefficient of determination of cross-validation (R2CV) was in fact ≥0.75). Excellent accuracy was observed for dry matter, protein, and S prediction in cross-validation and good prediction ability in external validation (R2CV ≥ 0.93; the coefficient of determination of external validation, R2V, was ≥0.82). Nonetheless, SCiO's ability to discriminate between good- and low-quality samples (IgG ≥ vs. < 50 g/L) was satisfactory. The affordable cost, the accurate predictions, and the user-friendly design, coupled with the increased interest in BC within the dairy sector, may boost the collection of extensive BC data for management and genetic purposes in the near future.

The effects of dietary iodine content, milking system, and farming practices on milk iodine concentration and quality traits.

Various management practices can influence milk quality traits in dairy cattle. As an example, an increasing investment in automatic milking system to substitute milking parlors has been observed in the last 2 decades in dairy farms which could have affected certain bulk milk quality traits. What is more, milking practices can also affect certain milk parameters; as an example, teat disinfectants containing I are used in commercial farms where pre- or postdipping is performed, leading to presence of some I in the bulk milk. However, this trace mineral is also supplied in cows' diet to fulfill their nutritional requirements, partly contributing to the milk I final concentration. Therefore, the aim of this study was to evaluate the sources of variation of milk I along with other traditional milk quality traits. A total of 91 dairy farms in northeastern Italy were enrolled in the study. In each farm, diet and bulk milk samples were collected on the same day for chemical analysis. Concentration of I, in particular, was determined in both milk and feed with gold standard. Pearson correlations were calculated among the traits available for milk and diet, and a general linear model was used to test significance of fixed effects (feeding system, milking system, farming system, herd size, herd stage of lactation, and sampling month) on milk quality traits including the I concentration. In the case of milk I, diet I and presence of I-based predipping and postdipping teat disinfect application were also tested as fixed effects. Results showed a positive linear correlation between milk and diet I content (correlation coefficient [r] = 0.78). Although milk I was also positively correlated with lactose content (r = 0.25), dietary I was not correlated with other milk traits. Milk I content was significantly affected by dietary I, I-based predipping teat disinfectant application, and herd composition. Compared with conventional farms, organic farms showed lower protein content and greater somatic cell score (SCS) but similar milk I. Milking system significantly affected only lactose content and SCS of milk. Sampling month was only significant for milk urea nitrogen and herd composition, feeding system, herd size, and herd average days in milk did not modify milk gross composition and SCS. In conclusion, dietary supply of I is the main factor affecting milk I concentration and findings suggest that I level in milk can be naturally improved in dairy cows by modulating the I content in the diet administered. However, further research is needed to evaluate the effect of I-based sanitizers on milk I.

Genetic evaluation of gestation length in Italian Holstein breed.

Gestation length (GL) can potentially affect health and performance of both the dam and the newborn calf, and it is controlled by two genetic components, direct and maternal. This means that both the calf (direct effect) and the cow (maternal effect) genotypes contribute to determine GL and its variability. The aims of the present study were to estimate direct and maternal variance components of GL, develop a routine genetic evaluation of GL in Italian Holstein and evaluate potential (un)favourable associations with traits for which selection is undertaken in this population. A multiple-trait repeatability linear animal model was employed for the estimation of variance components considering GL in first and later parities as different traits. The posterior mean (PM) of heritability of the direct effect was 0.43 for first parity and 0.35 for later parities. The PM of heritability of the maternal effect was lower, being 0.08 for primiparae and 0.06 for pluriparae. The posterior standard deviation (PSD) of the heritability estimates was small, ranging from 0.001 to 0.005. The relationship of direct and maternal effects with important traits such as milk yield and fertility indicated that selecting for extreme GL, longer or shorter, may have negative consequences on several traits, suggesting that GL has an intermediate optimum in dairy cattle. In conclusion, this study reveals that selecting an intermediate GL in the Italian Holstein population is advisable. Although scarcely variable compared to other conventional traits for which Italian Holstein is selected, GL is heritable and a deeper knowledge can be useful for decision-making at the farm level.

Teat morphology across five buffalo breeds: a multi-country collaborative study.

The buffalo (Bubalus bubalis) is a species of worldwide importance, raised to produce milk, meat, and hides, and often used as a working animal in rural contexts with low access to hi-tech solutions. In the present study, 100 lactating buffaloes (50 primiparous and 50 pluriparous) of five popular breeds were recruited to characterize and compare teat morphology. In particular, the focus was put on the Nili Ravi, Mediterranean, Egyptian, Bulgarian Murrah, and Azeri buffaloes raised in Pakistan, Italy, Egypt, Bulgaria, and Iran, respectively. In all countries, a longitudinal cross-section ultrasound was obtained before the milking to measure teat parameters at individual level: overall, teat canal length (TCL) averaged 24.13 mm, teat diameter (TD) 30.46 mm, cisternal diameter (CD) 17.80 mm, and teat wall (TW) 7.12 mm. The most variable trait across breeds was TCL which was positively correlated with CD and TD and negatively with TW, regardless of the teat position (front/rear or left/right). A strong negative correlation was found between TW and CD (- 0.43). The analysis of variance revealed that the fixed effect of breed significantly affected all the traits except TD. In fact, Bulgarian Murrah, Azeri, and Egyptian buffaloes presented the greatest estimate of TCL, whereas NR the smallest (14.70 mm). The TW was maximum in Nili Ravi, Egyptian, and Mediterranean buffaloes, with estimates equal to 8.19, 7.59, and 8.74 mm, respectively. Nili Ravi also showed the greatest TL (82.39 mm). In terms of CD, the lowest least square mean was that of Mediterranean buffaloes (12.14 mm). Primiparous and pluriparous buffaloes differed in terms of TD, TW, and TL, with older animals presenting the highest least square mean. In terms of position, instead, significant differences were observed for TD, CD, and TL when comparing front and rear teats, as left and right teats did not differ. Teat anatomy includes a set of heritable morphological features and is therefore breed-dependent. Differences presented in this study could be attributed to the divergent breeding objective and selective pressure across the five breeds; e.g., in some cases such as Mediterranean buffalo, selection for decades was oriented to improve milk production and milkability and achieve optimal conformation for mechanical milking. A better understanding of the mammary gland anatomical descriptors can be informative of the history of a breed and could provide useful insights to guide possible selection.

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples.

The renewable, abundant , and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples' microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited ß-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples. KEY POINTS: • Sequence- and function-based metagenomics were used to find CAZymes in compost samples. • Thermophilic composts proved to be rich in bacterial GH3, GH5, and GH9 enzymes. • Compost-derived fosmid libraries are enriched in clones with ß-glucosidase activity.

scFv biofunctionalized nanoparticles to effective and safe targeting of CEA-expressing colorectal cancer cells.

Colorectal cancer (CRC) is one of the deadliest cancers worldwide, with the 5 year survival rate in metastatic cases limited to 12%. The design of targeted and effective therapeutics remains a major unmet clinical need in CRC treatment. Carcinoembryonic antigen (CEA), a glycoprotein overexpressed in most colorectal tumors, may constitute a promising molecule for generating novel CEA-targeted therapeutic strategies for CRC treatment. Here, we developed a smart nanoplatform based on chemical conjugation of an anti-CEA single-chain variable fragment (scFv), MFE-23, with PLGA-PEG polymers to deliver the standard 5-Fluorouracil (5-FU) chemotherapy to CRC cells. We confirmed the specificity of the developed CEA-targeted NPs on the internalization by CEA-expressing CRC cells, with an enhance of threefold in the cell uptake. Additionally, CEA-targeted NPs loaded with 5-FU induced higher cytotoxicity in CEA-expressing cells, after 24 h and 48 h of treatment, reinforcing the specificity of the targeted NPs. Lastly, the safety of CEA-targeted NPs loaded with 5-FU was evaluated in donor-isolated macrophages, with no relevant impact on their metabolic activity nor polarization. Altogether, this proof of concept supports the CEA-mediated internalization of targeted NPs as a promising chemotherapeutic strategy for further investigation in different CEA-associated cancers and respective metastatic sites.Authors: Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 1 Given name: [Maria José] Last name [Silveira]. Author 7 Given name: [Maria José] Last name [Oliveira]. Also, kindly confirm the details in the metadata are correctokAffiliations: Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.ok.

Genetic characteristics of colostrum refractive index and its use as a proxy for the concentration of immunoglobulins in Holstein cattle.

BACKGROUND: Colostral concentration of immunoglobulins (Ig) is crucial for the passive transfer of antibodies from the cow to the new-born calf. Direct determination of Ig by the gold standard radial immunodiffusion method is demanding in terms of time and costs. For this reason, a refractometer is commonly used at the farm level for an indirect estimation of colostrum quality, which is given as the Ig concentration. In this study, colostrum samples were collected from 548 Italian Holstein cows within 6 h of calving. The refractive index (BRIX, %) of these samples was assessed using a portable optical refractometer, as well as the concentration of total protein, IgG, IgA, and IgM by radial immunodiffusion. A four-trait animal model was used to estimate genetic parameters for BRIX and the different immunoglobulin isotypes. A receiver operating characteristic analysis was carried out to evaluate the BRIX diagnostic accuracy. RESULTS: Colostral BRIX was moderately heritable (0.26) and its genetic and phenotypic correlations with IgG (0.91, 0.78), IgA (0.57, 0.57), and IgM (0.71, 0.61) were all positive and of similar order, although the genetic correlations were generally higher than the phenotypic correlations. Low-quality colostrum samples, defined as those with an IgG concentration lower than 50 g/L, were accurately identified by the refractive index on the BRIX scale, with an area under the curve of 0.90. CONCLUSIONS: The use of a refractometer is recommended on dairy farms to produce a proxy for colostral Ig concentration. BRIX is a useful phenotyping tool that can be used in cattle to improve the quality of colostrum for first feeding of calves through both traditional genetic and genomic strategies. Improving colostrum quality will reduce the incidence of failure of passive transfer of immunity in young stock.

Breeding for improved protein fractions and free amino acids concentration in bovine milk.

Considerable resources are required to routinely measure detailed milk compositional traits. Hence, an insufficient volume of phenotypic data can hinder genetic progress in these traits within dairy cow breeding programmes. The objective of the present study was to quantify the opportunities for breeding for improved milk protein and free amino acid (FAA) composition by exploiting mid-infrared spectroscopy (MIRS) predictions routinely recorded from milk samples. Genetic parameters for protein fractions and FAA composition were estimated using 134,546 test-day records from 16,166 lactations on 9,572 cows using linear mixed models. Heritability of MIRS-predicted protein fractions ranged from 0.19 (α-lactalbumin) to 0.55 (ß-lactoglobulin A), while heritability of MIRS-predicted FAA ranged from 0.08 for glycine to 0.29 for glutamic acid. Genetic correlations among the MIRS-predicted FAA were moderate to strong ranging from -0.44 (aspartic acid and lysine) to 0.97 (glutamic acid and total FAA). Adjustment of the genetic correlations for the genetic merit of 24-h milk yield did not greatly affect the correlations. Results from the current study highlight the presence of exploitable genetic variation for both protein fractions and FAA in dairy cow milk. Besides, the direction of genetic correlations reveals that breeding programmes directly selecting for greater milk protein concentration carry with them favourable improvement in casein and whey fractions.

Asymmetric Transfer Hydrogenation of Arylidene-Substituted Chromanones and Tetralones Catalyzed by Noyori-Ikariya Ru(II) Complexes: One-Pot Reduction of C═C and C═O bonds.

3-Arylidenechroman-4-ones and 2-arylidene-1-tetralones are hydrogenated to cis-benzylic alcohols in dr's and er's up to 99:1 via a C═C and C═O one-pot reduction in the presence of 2-5 mol % Noyori-Ikariya-type RuII chiral complexes and HCO2Na as a hydrogen source under asymmetric transfer hydrogenation-dynamic kinetic resolution (ATH-DKR) conditions. The oxidation of theses substrates resulted in the enantioselective synthesis of the natural homoisoflavanone dihydrobonducellin and its carba-analogues.

The T-box transcription factor brachyury behaves as a tumor suppressor in gliomas.

The oncogene brachyury (TBXT) is a T-box transcription factor that is overexpressed in multiple solid tumors and is associated with tumor aggressiveness and poor patient prognosis. Gliomas comprise the most common and aggressive group of brain tumors, and at the present time the functional and clinical impact of brachyury expression has not been investigated previously in these neoplasms. Brachyury expression (mRNA and protein) was assessed in normal brain (n = 67), glioma tissues (n = 716) and cell lines (n = 42), and further in silico studies were undertaken using genomic databases totaling 3115 samples. Our glioma samples were analyzed for copy number (n = 372), promoter methylation status (n = 170), and mutation status (n = 1569 tissues and n = 52 cell lines) of the brachyury gene. The prognostic impact of brachyury expression was studied in 1524 glioma patient tumors. The functional impact of brachyury on glioma proliferation, viability, and cell death was evaluated both in vitro and in vivo. Brachyury was expressed in the normal brain, and significantly downregulated in glioma tissues. Loss of brachyury was associated with tumor aggressiveness and poor survival in glioma patients. Downregulation of brachyury was not associated with gene deletion, promoter methylation, or inactivating point mutations. Brachyury re-expression in glioma cells was found to decrease glioma tumorigenesis by induction of autophagy. These data strongly suggest that brachyury behaves as a tumor suppressor gene in gliomas by modulating autophagy. It is important to note that brachyury constitutes an independent positive biomarker of patient prognosis. Our findings indicate that the role of brachyury in tumorigenesis may be tissue-dependent and demands additional investigation to guide rational interventions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The concentrations of immunoglobulins in bovine colostrum determined by the gold standard method are genetically correlated with their near-infrared prediction.

BACKGROUND: The quality of colostrum administered to calves is based on its concentration in immunoglobulins G (IgG, g/L). Immunoglobulins A (IgA) and M (IgM) are also present but at a lower level. The gold standard reference analysis for these traits, radial immunodiffusion, is time-consuming and expensive. In order to define breeding strategies that are aimed at improving colostrum quality in dairy cattle, a large amount of data is needed, and the use of indicator traits would be beneficial. In the study presented here, we explored the heritabilities of reference (radial immunodiffusion) and near infrared-predicted IgG, IgA, and IgM concentrations and estimated their genetic correlations. First, the colostrum of 765 Holstein cows from nine herds was sampled to perform a reference analysis and the near-infrared spectra (400-2500 nm) were stored. We used a calibration set (28% of the initial samples) that was representative of the herds and cow parity orders to develop prediction equations for IgG, IgA, and IgM concentrations. Finally, these traits were predicted in the validation set (72% of the initial samples) to estimate genetic parameters for the predictions. Genetic correlations between reference and predicted values of each trait were estimated through bivariate linear animal models. RESULTS: The three near-infrared-predicted immunoglobulin fractions were genetically correlated with their reference value. In particular, the reference and predicted IgG concentrations were strongly correlated at both the genetic (0.854 ± 0.314) and phenotypic level (0.767 ± 0.019). Weaker associations were observed for IgA and IgM concentrations, which were predicted with lower accuracy compared to IgG. Simulation analyses suggested that improving colostrum quality by selective breeding in Holstein cattle based on near-infrared predicted colostrum immunoglobulins concentrations is feasible. In addition, less than 10 mL of colostrum are needed for spectra acquisition and thus implementation of such analyses is possible in the near future. CONCLUSIONS: The concentrations of colostrum immunoglobulins can be predicted from near-infrared spectra and the genetic correlation between the reference and the predicted traits is positive and favourable, in spite of the large standard errors of the estimates. Near-infrared spectroscopy can be exploited in selective breeding of dairy cattle to improve colostral immunoglobulins concentration.

Synthesis of new α-Aryl-α-tetralones and α-Fluoro-α-aryl-α-tetralones, preliminary antiproliferative evaluation on drug resistant cell lines and in silico prediction of ADMETox properties.

α-aryl-α-tetralones and α-fluoro-α-aryl-α-tetralones derivatives were synthesized by palladium catalyzed α-arylation reaction of α-tetralones and α-fluoro-α-tetralones, with bromoarenes in moderate to good yields. These compounds were evaluated for their in vitro anti-proliferative effects against human breast cancer and leukemia cell lines with diverse profiles of drug resistance. The most promising compounds, 3b, 3c, 8a and 8c, were effective on both neoplastic models. 3b and 8a induced higher toxicity on multidrug resistant cells and were able to avoid efflux by ABCB1 and ABCC1 transporters. Theoretical calculations of the physicochemical descriptors to predict ADMETox properties were favorable concerning Lipinski's rule of five, results that reflected on the low effects on non-tumor cells. Therefore, these compounds showed great potential for development of pharmaceutical agents against therapy refractory cancers.

Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies.

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (> 99% of humic acids effectively removed) and sufficient quantity (10.5-13.8 µg g-1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory. KEY POINTS/HIGHLIGHTS: • Metagenomic DNA was successfully extracted from compost samples rich in humic acids • The improved protocol was established by optimizing the cell lysis method and buffer • Complete removal of humic acids was achieved through the use of activated charcoal • The suitability of the DNA was proven by the construction of a metagenomic library.

Euphol, a tetracyclic triterpene, from Euphorbia tirucalli induces autophagy and sensitizes temozolomide cytotoxicity on glioblastoma cells.

Glioblastoma (GBM) is the most frequent and aggressive type of brain tumor. There are limited therapeutic options for GBM so that new and effective agents are urgently needed. Euphol is a tetracyclic triterpene alcohol, and it is the main constituent of the sap of the medicinal plant Euphorbia tirucalli. We previously identified anti-cancer activity in euphol based on the cytotoxicity screening of 73 human cancer cells. We now expand the toxicological screening of the inhibitory effect and bioactivity of euphol using two additional glioma primary cultures. Euphol exposure showed similar cytotoxicity against primary glioma cultures compared to commercial glioma cells. Euphol has concentration-dependent cytotoxic effects on cancer cell lines, with more than a five-fold difference in the IC50 values in some cell lines. Euphol treatment had a higher selective cytotoxicity index (0.64-3.36) than temozolomide (0.11-1.13) and reduced both proliferation and cell motility. However, no effect was found on cell cycle distribution, invasion and colony formation. Importantly, the expression of the autophagy-associated protein LC3-II and acidic vesicular organelle formation were markedly increased, with Bafilomycin A1 potentiating cytotoxicity. Finally, euphol also exhibited antitumoral and antiangiogenic activity in vivo, using the chicken chorioallantoic membrane assay, with synergistic temozolomide interactions in most cell lines. In conclusion, euphol exerted in vitro and in vivo cytotoxicity against glioma cells, through several cancer pathways, including the activation of autophagy-associated cell death. These findings provide experimental support for further development of euphol as a novel therapeutic agent for GBM, either alone or in combination chemotherapy.

On the genomic regions associated with milk lactose in Fleckvieh cattle.

Lactose is a sugar uniquely found in mammals' milk and it is the major milk solid in bovines. Lactose yield (LY, kg/d) is responsible for milk volume, whereas lactose percentage (LP) is thought to be more related to epithelial integrity and thus to udder health. There is a paucity of studies that have investigated lactose at the genomic level in dairy cows. This paper aimed to improve our knowledge on LP and LY, providing new insights into the significant genomic regions affecting these traits. A genome-wide association study for LP and LY was carried out in Fleckvieh cattle by using bulls' deregressed estimated breeding values of first lactation as pseudo-phenotypes. Heritabilities of first-lactation test-day LP and LY estimated using linear animal models were 0.38 and 0.25, respectively. A total of 2,854 bulls genotyped with a 54K SNP chip were available for the genome-wide association study; a linear mixed model approach was adopted for the analysis. The significant SNP of LP were scattered across the whole genome, with signals on chromosomes 1, 2, 3, 7, 12, 16, 18, 19, 20, 28, and 29; the top 4 significant SNP explained 4.90% of the LP genetic variance. The signals were mostly in regions or genes with involvement in molecular intra- or extracellular transport; for example, CDH5, RASGEF1C, ABCA6, and SLC35F3. A significant region within chromosome 20 was previously shown to affect mastitis or somatic cell score in cattle. As regards LY, the significant SNP were concentrated in fewer regions (chromosomes 6 and 14), related to mastitis/somatic cell score, immune response, and transport mechanisms. The 5 most significant SNP for LY explained 8.45% of genetic variance and more than one-quarter of this value has to be attributed to the variant within ADGRB1. Significant peaks in target regions remained even after adjustment for the 2 most significant variants previously detected on BTA6 and BTA14. The present study is a prelude for deeper investigations into the biological role of lactose for milk secretion and volume determination, stressing the connection with genes regulating intra- or extracellular trafficking and immune and inflammatory responses in dairy cows. Also, these results improve the knowledge on the relationship between lactose and udder health; they support the idea that LP and its derived traits are potential candidates as indicators of udder health in breeding programs aimed to enhance cows' resistance to mastitis.

Insecticidal activity of indole derivatives against Plutella xylostella and selectivity to four non-target organisms.

The diamondback moth Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae) is a destructive pest of brassica crops of economic importance that have resistance to a range of insecticides. Indole derivates can exert diverse biological activities, and different effects may be obtained from small differences in their molecular structures. Indole is the parent substance of a large number of synthetic and natural compounds, such as plant and animal hormones. In the present study, we evaluate the insecticidal activity of 20 new synthesized indole derivatives against P. xylostella, and the selectivity of these derivatives against non-target hymenopteran beneficial arthropods: the pollinator Apis mellifera (Linnaeus, 1758) (Hymenoptera: Apidae), and the predators Polybia scutellaris (White, 1841), Polybia sericea (Olivier, 1791) and Polybia rejecta (Fabricius, 1798) (Hymenoptera: Vespidae). Bioassays were performed in the laboratory to determine the lethal and sublethal effects of the compounds on P. xylostella and to examine their selectivity to non-target organisms by topical application and foliar contact. The treatments consisted of two synthesized derivatives (most and least toxic), the positive control (deltamethrin) and the negative control (solvent). The synthesized compound 4e [1-(1H-indol-3-yl)hexan-1-one] showed high toxicity (via topical application and ingestion) and decreased the leaf consumption by P. xylostella, displaying a higher efficiency than the pyrethroid deltamethrin, widely used to control this pest. In addition, the synthesized indole derivatives were selective to the pollinator A. mellifera and the predators P. scutellaris, P. sericea and P. rejecta, none of which were affected by deltamethrin. Our results highlight the promising potential of the synthesized indole derivatives for the generation of new chemical compounds for P. xylostella management.

Cross reactivity of commercial anti-dengue immunoassays in patients with acute Zika virus infection.

Several countries have local transmission of multiple arboviruses, in particular, dengue and Zika viruses, which have recently spread through many American countries. Cross reactivity among Flaviviruses is high and present a challenge for accurate identification of the infecting agent. Thus, we evaluated the level of cross reactivity of anti-dengue IgM/G Enzyme-Linked Immunosorbent Assays (ELISA) from three manufacturers against 122 serum samples obtained at two time-points from 61 patients with non-dengue confirmed Zika virus infection. All anti-dengue ELISAs cross reacted with serum from patients with acute Zika infection at some level and a worrisome number of seroconversion for dengue IgG and IgM was observed. These findings may impact the interpretation of currently standard criteria for dengue diagnosis in endemic regions.