Penicillium chrysogenum as a fungal factory for feruloyl esterases.

Plant biomass is a promising substrate for biorefinery, as well as a source of bioactive compounds, platform chemicals, and precursors with multiple industrial applications. These applications depend on the hydrolysis of its recalcitrant structure. However, the effective biological degradation of plant cell walls requires several enzymatic groups acting synergistically, and novel enzymes are needed in order to achieve profitable industrial hydrolysis processes. In the present work, a feruloyl esterase (FAE) activity screening of Penicillium spp. strains revealed a promising candidate (Penicillium rubens Wisconsin 54-1255; previously Penicillium chrysogenum), where two FAE-ORFs were identified and subsequently overexpressed. Enzyme extracts were analyzed, confirming the presence of FAE activity in the respective gene products (PrFaeA and PrFaeB). PrFaeB-enriched enzyme extracts were used to determine the FAE activity optima (pH 5.0 and 50-55 °C) and perform proteome analysis by means of MALDI-TOF/TOF mass spectrometry. The studies were completed with the determination of other lignocellulolytic activities, an untargeted metabolite analysis, and upscaled FAE production in stirred tank reactors. The findings described in this work present P. rubens as a promising lignocellulolytic enzyme producer. KEY POINTS: • Two Penicillium rubens ORFs were first confirmed to have feruloyl esterase activity. • Overexpression of the ORFs produced a novel P. rubens strain with improved activity. • The first in-depth proteomic study of a P. rubens lignocellulolytic extract is shown.

Use of intrapartum ultrasound in term pregnant women with contractions before hospital admission.

INTRODUCTION: The aim of this study was to test the hypothesis that transperineal ultrasound can be used to decide whether to admit a pregnant woman due to labor. MATERIAL AND METHODS: In this analytical cross-sectional observational study, transperineal ultrasound was performed on pregnant women with intact membranes who came to the hospital due to contractions. A decision was made to admit women due to labor based on the ultrasound measurements. The ultrasound measurements were used to determine cervical dilation, the angle of progression, and fetal head position. The managing midwives were blinded to the results and made the final decision to admit the women based on digital vaginal examination. RESULTS: It was possible to decide whether a woman had to be admitted for delivery or discharged due to the latent phase of labor according to the ultrasound examination in 55 of the 57 cases (96.5%). In four of the 55 cases, the decision based on ultrasound differed from the midwife's decision (7.3%). There was strong agreement between the decision to admit the pregnant women based on ultrasound measurements and the digital vaginal examination (Cohen's kappa: 0.844). It was possible to measure cervical dilation with ultrasound in 52 of the 57 cases (91.2%). The intraclass correlation coefficient for the cervical dilation measurements was 0.736 (95% confidence interval 0.539-0.848). CONCLUSIONS: There was strong agreement between the ultrasound and digital vaginal examination results in the decision to admit singleton pregnant women at term due to labor. A large number of vaginal examinations could be avoided by using intrapartum ultrasound.

Diagnostics of DNA fragmentation in human spermatozoa: Are sperm chromatin structure analysis and sperm chromatin dispersion tests (SCD-HaloSpermG2® ) comparable?

Men affected with idiopathic infertility often display basic spermiogramme values similar to fertile individuals, questioning the diagnostic impact of the World Health Organization (WHO) thresholds used. This study explored sperm DNA fragmentation in single ejaculates from 14 fertile donors and 42 patients with idiopathic infertility providing semen for assisted reproductive techniques in a university fertility clinic. Each ejaculate was simultaneously studied for sperm DNA fragmentation by the flow cytometer-based sperm chromatin structure analysis (SCSA) and the new light-microscopy-based sperm chromatin dispersion assay (SCD-HaloSpermG2® ), before and after sperm selection for in vitro fertilisation with a colloid discontinuous gradient. The WHO semen variables did not differ between groups, but DNA fragmentation after SCSA (DFI) or SCD (SDF) was significantly (p < 0.05) higher in patients (DFI: 40.2% ± 3.0 vs. SDF: 40.3% ± 1.4) than in fertile donors (DFI: 17.1% ± 2.1 vs. SDF: 20.9% ± 2.5). Sperm selection led to lower proportions of DNA-fragmented spermatozoa (DFI: 11.9 ± 1.7 vs. SCD: 10.0 ± 0.9, p < 0.05). The techniques output correlated highly and significantly (r2  = 0.82). DNA fragmentation is confirmed as a relevant variable for scrutinising patients with idiopathic infertility, beyond the evidently insufficient WHO semen analyses. Since both techniques yielded similar results, the reduced necessity of complex equipment when running SCD ought to be considered for a clinical setting.

Changes in Cell Wall Polymers and Degradability in Maize Mutants Lacking 3'- and 5'-O-Methyltransferases Involved in Lignin Biosynthesis.

Caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) and caffeic acid-O-methyltransferase (COMT) are key enzymes in the biosynthesis of coniferyl and sinapyl alcohols, the precursors of guaiacyl (G) and syringyl (S) lignin subunits. The function of these enzymes was characterized in single and double mutant maize plants. In this work, we determined that the comt (brown-midrib 3) mutant plants display a reduction of the flavonolignin unit derived from tricin (a dimethylated flavone), demonstrating that COMT is a key enzyme involved in the synthesis of this compound. In contrast, the ccoaomt1 mutants display a wild-type amount of tricin, suggesting that CCoAOMT1 is not essential for the synthesis of this compound. Based on our data, we suggest that CCoAOMT1 is involved in lignin biosynthesis at least in midribs. The phenotype of ccoaomt1 mutant plants displays no alterations, and their lignin content and composition remain unchanged. On the other hand, the ccoaomt1 comt mutant displays phenotypic and lignin alterations similar to those already described for the comt mutant. Although stems from the three mutants display a similar increase of hemicelluloses, the effect on cell wall degradability varies, the cell walls of ccoaomt1 being the most degradable. This suggests that the positive effect of lignin reduction on cell wall degradability of comt and ccoaomt1 comt mutants is counteracted by changes occurring in lignin composition, such as the decreased S/G ratio. In addition, the role of the flavonolignin unit derived from tricin in cell wall degradability is also discussed.

Optimized Fast Filtration-Based Sampling and Extraction Enables Precise and Absolute Quantification of the Escherichia coli Central Carbon Metabolome.

Precise and accurate quantification is a prerequisite for interpretation of targeted metabolomics data, but this task is challenged by the inherent instability of the analytes. The sampling, quenching, extraction, and sample purification conditions required to recover and stabilize metabolites in representative extracts have also been proven highly dependent on species-specific properties. For Escherichia coli, unspecific leakage has been demonstrated for conventional microbial metabolomics sampling protocols. We herein present a fast filtration-based sampling protocol for this widely applied model organism, focusing on pitfalls such as inefficient filtration, selective loss of biomass, matrix contamination, and membrane permeabilization and leakage. We evaluate the effect of and need for removal of extracellular components and demonstrate how residual salts can challenge analytical accuracy of hyphenated mass spectrometric analyses, even when sophisticated correction strategies are applied. Laborious extraction procedures are bypassed by direct extraction in cold acetonitrile:water:methanol (3:5:2, v/v%), ensuring compatibility with sample concentration and thus, any downstream analysis. By applying this protocol, we achieve and demonstrate high precision and low metabolite turnover, and, followingly, minimal perturbation of the inherent metabolic state. This allows us to herein report absolute intracellular concentrations in E. coli and explore its central carbon metabolome at several commonly applied cultivation conditions.

Effect of Honey, Coenzyme Q10, and ß-Carotene/α-Tocopherol as Novel Additives in Rabbit-Sperm Cryopreservation Extender.

The effectiveness of rabbit-sperm cryopreservation is still below average compared to other domestic species. After the sperm cryopreservation process, post-thawing parameters like motility and membrane integrity are significantly compromised. The use of new extender constituents is an approach that can be used to improve the effectiveness of cryopreservation. Accordingly, we used honey (1.25, 2.5, 5, and 10%), coenzyme Q10 (100 and 200 µM), and ß-carotene/α-tocopherol (500 µM/620 µM and 250 µM/310 µM) as candidate components for rabbit-sperm extenders during cryopreservation. Ejaculates from commercial adult rabbit bucks (n = 5) were cryopreserved using conventional freezing. Several post-thawing sperm parameters were assessed, including total motility, membrane integrity, viability, nuclear membrane integrity, acrosome reaction, and mitochondrial membrane potential and activation. Additionally, we performed hormonal analyses of the seminal plasma. Moreover, we analyzed the post-thawing levels of a molecular marker of sperm quality, proAKAP4, which was used in rabbits for the first time. Our findings showed that the 2.5% honey supplementation increased the post-thawing sperm motility (13.75 ± 3.75%) compared to the greater concentrations employed. However, the post-thawing motility was negatively affected by the coenzyme Q10 (0%, in both groups) but was not affected by the ß-carotene/α-tocopherol supplementation (22 ± 18.15%, and 11.67 ± 10.17%). In conclusion, the cryopreservation protocols of this study did not help to maintain the sperm parameters after thawing. Further studies are required to identify novel protocols to mitigate the damage caused to rabbit sperm during cryopreservation.

Sentinel Node Biopsy in Patients With Breast Carcinoma Without Axillary Involvement at Diagnosis Receiving Primary Systemic Therapy.

BACKGROUND: Primary systemic therapy (PST) has acquired great importance in breast cancer (BC) in the last few years. In this scenario, even if it is accepted to perform SLNB before PST, most of the guidelines remark the advantages of this practice after it, such as avoiding another surgery to the patient, a rapid start of the treatment and no need of axillary dissection in cases of pathologic complete response (pCR). Nevertheless, the lack of knowledge of the initial axillary state and the need to practice axillary dissection with any axillary disease are claimed to be some other disadvantages. There are no randomized studies yet that can conclude the optimal timing of SLNB in PST, so for the moment we may settle for our common practice. PATIENTS AND METHODS: We studied all the cases attended in the Breast Unit that joined the inclusion criteria between 2011 and 2019 in our hospital and we compared the group with SLNB before PST with the group with SLNB after PST in terms of unnecessary axillary dissection and description features. RESULTS: We included 223 female patients diagnosed with BC and without clinical nor radiological axillary disease (cN0), who had received NAC and SLNB performed before or after it. We observed a higher proportion of high-grade histological tumors (G3), tumors with aggressive phenotypes (Basal like and Her 2 enriched), and younger women in the group of SLNB before NAC compared with the SLNB after NAC group (P < .01). Despite this, we did not find any difference in the number of positive SLNBs or in the number of ALND performed between the 2 groups. We found a higher proportion of ALND with all the lymph node (LN) negatives in the SLNB before NAC group. CONCLUSION: Taking into account that in the observation period we did not use ACOSOG Z0011 criteria with all the SLNBs, we figure out what would have been the real results nowadays following these criteria. In this scenario we conclude that patients with luminal phenotype seemed to benefit from practicing SLNB before NAC in terms of avoiding axillary dissections. We could not make any conclusion in the rest of the phenotypes. However, prospective studies are needed to confirm if this affirmation could be proved.

Experimental determination of Escherichia coli biomass composition for constraint-based metabolic modeling.

Genome-scale metabolic models (GEMs) are mathematical representations of metabolism that allow for in silico simulation of metabolic phenotypes and capabilities. A prerequisite for these predictions is an accurate representation of the biomolecular composition of the cell necessary for replication and growth, implemented in GEMs as the so-called biomass objective function (BOF). The BOF contains the metabolic precursors required for synthesis of the cellular macro- and micromolecular constituents (e.g. protein, RNA, DNA), and its composition is highly dependent on the particular organism, strain, and growth condition. Despite its critical role, the BOF is rarely constructed using specific measurements of the modeled organism, drawing the validity of this approach into question. Thus, there is a need to establish robust and reliable protocols for experimental condition-specific biomass determination. Here, we address this challenge by presenting a general pipeline for biomass quantification, evaluating its performance on Escherichia coli K-12 MG1655 sampled during balanced exponential growth under controlled conditions in a batch-fermentor set-up. We significantly improve both the coverage and molecular resolution compared to previously published workflows, quantifying 91.6% of the biomass. Our measurements display great correspondence with previously reported measurements, and we were also able to detect subtle characteristics specific to the particular E. coli strain. Using the modified E. coli GEM iML1515a, we compare the feasible flux ranges of our experimentally determined BOF with the original BOF, finding that the changes in BOF coefficients considerably affect the attainable fluxes at the genome-scale.

Microbial Isolation and Characterization of New Antibiotic-Producing Strains from Decayed Wood.

Microbial drug resistance is increasing over the last years, becoming one of the most important health concerns in the twenty-first century. It encourages the discovery of new antibiotics. Thus, novel antibiotics discovered by exploring different environments that previously have been left out of the scientific focus is a realistic opportunity. One of these habitats can be forest deadwood, which is a specific niche inside of the forest that provides shelter and nutrition to a great variety of organisms, such as fungi, bacteria, or saproxylic insects. Different studies have found the existence of complex antagonisms and symbiotic interactions among them, which points at decayed wood as a competitive environment. Besides, it is an interesting niche to look for new antibiotic producer microorganisms and active chemicals. This chapter describes isolation and screening methods of novel producers of antimicrobial compounds from decayed wood.

Screening of Antibiotic Gene Clusters in Microorganisms Isolated from Wood.

The biosphere of Earth is made up of a variety of ecosystems governed by complex biological interactions, some of them mediated by microbial bioactive secondary metabolites. These metabolites such as antibiotics (e.g., polyketides and nonribosomal peptides) have been receiving increasing attention, due to their multiple pharmaceutical uses. Besides, antibiotic resistance is on the rise, and it is currently regarded as one of the greatest threats to global human health. The screening of novel antimicrobial polyketides and nonribosomal peptides in poorly studied ecosystems is an interesting alternative to address the problem of antibiotic resistance. This chapter updates a molecular method to identify antibiotics gene clusters and their subsequent production and activity validation. On the one hand, a PCR method based on degenerated primers for nonribosomal peptide synthases (NRPS) and the polyketide synthases (PKS) genes is used as an initial fast screening. On the other hand, a bioassay-based method is the protocol selected for the production confirmation and antibacterial effect estimation. These methods are applied to screen Actinobacteria and Penicillium species as main antibiotic producers isolated from wood.

Synthesis and anti-leishmanial evaluation of 1-phenyl-2,3,4,9-tetrahydro-1H-ß-carboline derivatives against Leishmania infantum.

In the present study, antileishmanial activity of sixteen novel series of tetrahydro-ß-carboline derivatives against transgenic infrared fluorescent Leishmania infantum strain has been reported. Among these reported analogues, most of the compounds exhibited potent inhibition against both promastigote (IC50 from 1.99 ± 1.40 to 20.69 ± 0.95 µM) and amastigote (IC50 from 0.67 ± 0.05 to 4.16 ± 0.008 µM) forms of L. infantum. Moreover, compound 7l, displayed most potent and selective inhibition of parasite amastigote form with IC50 0.67 ± 0.05 µM, selectivity index >298.5 and was comparable with standard drug amphotericin B. From this study, a new class of tetrahydro-ß-carboline derivatives with potent antileishmanial activity was identified and it needs further extensive study to optimize the lead molecules to win the battle against severe and neglected disease leishmaniasis.

Cell wall modifications triggered by the down-regulation of Coumarate 3-hydroxylase-1 in maize.

Coumarate 3-hydroxylase (C3H) catalyzes a key step of the synthesis of the two main lignin subunits, guaiacyl (G) and syringyl (S) in dicotyledonous species. As no functional data are available in regards to this enzyme in monocotyledonous species, we generated C3H1 knock-down maize plants. The results obtained indicate that C3H1 participates in lignin biosynthesis as its down-regulation redirects the phenylpropanoid flux: as a result, increased amounts of p-hydroxyphenyl (H) units, lignin-associated ferulates and the flavone tricin were detected in transgenic stems cell walls. Altogether, these changes make stem cell walls more degradable in the most C3H1-repressed plants, despite their unaltered polysaccharide content. The increase in H monomers is moderate compared to C3H deficient Arabidopsis and alfalfa plants. This could be due to the existence of a second maize C3H protein (C3H2) that can compensate the reduced levels of C3H1 in these C3H1-RNAi maize plants. The reduced expression of C3H1 alters the macroscopic phenotype of the plants, whose growth is inhibited proportionally to the extent of C3H1 repression. Finally, the down-regulation of C3H1 also increases the synthesis of flavonoids, leading to the accumulation of anthocyanins in transgenic leaves.

Plasmocitoma mamario y cáncer de mama: a propósito de un caso / Breast Plasmacytoma and breast cancer: a case report

El plasmocitoma mamario es una neoplasia de células plasmáticas extremadamente infrecuente, con menos de cincuenta casos descritos en el último siglo. Por este motivo, apenas se dispone de datos acerca del abordaje, tratamiento y seguimiento más convenientes. Presentamos el caso de una paciente de 70 años que debutó con un plasmocitoma mamario y que un año después fue diagnosticada de un carcinoma mamario lobulillar ipsilateral. La asociación entre plasmocitoma y cáncer de mama no está descrita en la literatura, por lo que es muy complicado establecer un vínculo entre ambas entidades. Sin embargo, el abordaje terapéutico del plasmocitoma podría comprometer el tratamiento ulterior de un cáncer de mama, por lo que el tratamiento idóneo en estos casos sea probablemente la cirugía.

Breast plasmocytoma is an extremely rare plasma cell neoplasm, with less than 50 cases reported in the last century. This is the reason why we barely have data about optimal management, treatment and follow-up. We hereby report the case of a 70 year old woman diagnosed with breast plasmocytoma that developed lobular breast cancer a year later. The link between plasmocytoma and breast cancer has not been previously established. However, breast plasmocytoma treatment could compromise latter breast cancer approach, so probably the most suitable strategy in these cases should be breast surgery.Conclusions: There are clinical characteristics associated with complications in women with surgical management abortion in our center, such as admission diagnosis, unplanned pregnancy, previous abortion and type of evacuation. There are limitations regarding the quantity and quality of information, however, our results allow us to know the profile of patients treated for abortion in our center.

Fourier transform mid infrared spectroscopy applications for monitoring the structural plasticity of plant cell walls.

Fourier transform mid-infrared (FT-MIR) spectroscopy has been extensively used as a potent, fast and non-destructive procedure for analyzing cell wall architectures, with the capacity to provide abundant information about their polymers, functional groups, and in muro entanglement. In conjunction with multivariate analyses, this method has proved to be a valuable tool for tracking alterations in cell walls. The present review examines recent progress in the use of FT-MIR spectroscopy to monitor cell wall changes occurring in muro as a result of various factors, such as growth and development processes, genetic modifications, exposition or habituation to cellulose biosynthesis inhibitors and responses to other abiotic or biotic stresses, as well as its biotechnological applications.