Nuria Estévez-Gómez, Tamara Prieto, Laura Tomás
et al.
Summary: Single-cell genomics enables studying tissues and organisms at the highest resolution. However, since a cell contains a small amount of DNA, single-cell DNA sequencing (scDNA-seq) typically requires single-cell whole-genome amplification (scWGA). Unfortunately, scWGA methods introduce technical biases that complicate the interpretation of scDNA-seq data. We compared six scWGA methods, three MDA (multiple displacement amplification; GenomiPhi, REPLI-g, and TruePrime) and three non-MDA (Ampli1, MALBAC, and PicoPLEX), on 206 tumoral and 24 healthy human cells. scWGA methods performed differently depending on the parameter of interest. REPLI-g minimized regional amplification bias, while non-MDA methods showed a more uniform and reproducible amplification. Ampli1 exhibited the lowest allelic imbalance and dropout, the most accurate insertion or deletion (indel) and copy-number detection, and a low polymerase error rate. However, REPLI-g yielded higher DNA quantities, longer amplicons, and greater genome coverage. We offer a comprehensive guide for selecting a scWGA approach, outlining trade-offs that influence the interpretation of scDNA-seq data. Motivation: Single-cell whole-genome amplification (scWGA) is a critical step for the genomic study of single cells, yet its impact on downstream analysis remains poorly understood. Previous studies were not comprehensive in the number of methods tested and the metrics assessed. Besides, they have mostly been conducted or replicated by the original authors of a method rather than by independent third-party laboratories. Here, we test six commercially available scWGA methods to understand their performance and propose a guide for selecting the best scWGA approach for studies on single-cell genomics.
Abstract Fragaria iinumae, a diploid progenitor species of octoploid strawberries, likely occupies a basal position within the genus Fragaria. In this study, we report a near-complete genome assembly of F. iinumae v2.0, totaling 241.14 Mb with a contig N50 of 33.31 Mb. We identified 14 telomeric and 7 centromeric regions across its seven chromosomes. Compared to previous assemblies, F. iinumae v2.0 demonstrates substantial improvements in both genome continuity (gaps reduced from 29 to 0) and annotation completeness, including the annotation of 4,144 new genes and 395 new gene clusters. Notably, several large structural variants were identified between the F. iinumae v1.0 and F. iinumae v2.0 genomes, with most gaps in the v1.0 assembly overlapping with structural variant breakpoints. Additionally, we found a significant expansion of telomeric repeats in the B subgenome of octoploid strawberries compared to F. iinumae. Interestingly, two telomeres consistently exhibited low repeat abundance in both the diploid and octoploid B subgenomes, suggesting significant contraction early in the evolution of F. iinumae. Furthermore, through multiple lines of genomic evidence—including phylogenetic analyses, genetic distance matrices, a burst of LTR insertions, and the distribution of NLR resistance genes, we conclude that F. iinumae may represent an early-diverging lineage within the strawberry genus. This updated assembly provides a crucial genomic resource for understanding of the origin and structural dynamics of the strawberry genus and facilitates further exploration of genome-wide consequences of polyploidy.
Clemens Steinek, Miguel Guirao-Ortiz, Gabriela Stumberger
et al.
Summary: The genome contains numerous regulatory elements that may undergo complex interactions and contribute to the establishment, maintenance, and change of cellular identity. Three-dimensional genome organization can be explored with fluorescence in situ hybridization (FISH) at the single-cell level, but the detection of small genomic loci remains challenging. Here, we provide a rapid and simple protocol for the generation of bright FISH probes suited for the detection of small genomic elements. We systematically optimized probe design and synthesis, screened polymerases for their ability to incorporate dye-labeled nucleotides, and streamlined purification conditions to yield nanoscopy-compatible oligonucleotides with dyes in variable arrays (NOVA probes). With these probes, we detect genomic loci ranging from genome-wide repetitive regions down to non-repetitive loci below the kilobase scale. In conclusion, we introduce a simple workflow to generate densely labeled oligonucleotide pools that facilitate detection and nanoscopic measurements of small genomic elements in single cells. Motivation: While three-dimensional chromatin conformations can be explored with fluorescence in situ hybridization (FISH), the visualization of small genomic loci with high spatial resolution remains challenging. For such applications, programmable oligonucleotides with high brightness are required. To further improve precision and sensitivity, secondary hybridization steps should be omitted. Here, we present a simple, quick, and inexpensive approach to generate labeled FISH probes that carry several fluorophores. Our workflow allows for the free choice of fluorophores, flexible adjustment of labeling density, and selective probe synthesis from large probe pools. With our probes, we reliably detect genomic loci below the kilobase level and examine their topological relationships.
After maturity, seed vigor irreversibly decreases. Understanding the underlying mechanism is important to germplasm preservation. MicroRNAs (miRNAs) play vital regulatory roles in plants. However, little is known about how miRNAs regulate seed aging. Here, elm (<i>Ulmus pumila</i> L.) seeds of three aging stages were subjected to a multi-omics analysis including transcriptome, small RNAome and degradome, to find regulators of seed aging. In the small RNAome, 119 miRNAs were identified, including 111 conservative miRNAs and eight novel miRNAs specific to elm seeds, named upu-miRn1-8. A total of 4900 differentially expressed genes, 22 differentially expressed miRNAs, and 528 miRNA-target pairs were identified during seed ageing. The target genes were mainly involved in the processing of proteins in the endoplasmic reticulum, metabolism, plant hormone signal transduction, and spliceosome. The expression of several DEGs and miRNAs were verified by qRT-PCR. The degradome data showed the exact degradation sites of upu-miR399a on <i>ABCG25</i>, and upu-miR414a on <i>GIF1</i>, etc. The dual-luciferase assay verified the negative regulation of upu-miR399a on <i>ABCG25</i> and upu-miR414a on <i>GIF1</i> in tobacco leaves. This study outlined the regulation network of mRNA, miRNA and miRNA-target genes during seed aging, which is helpful in integrating the regulation mechanisms of seed vigor at the transcriptional and post-transcriptional levels.
José Escurra, Francisco P. Ferreira, Tomás R. López
et al.
Lactic acid is a compound used industrially due to its properties. There are two methods for its production: chemical synthesis and microbial fermentation. In microbial fermentation, food industry waste can be used as a substrate, providing a route towards achieving a circular economy. Thus, this study evaluated different substrates for <i>Lactobacillus plantarum</i> growth, a lactic acid producer, such as molasses, whey, glucose, and saccharose, either alone or supplemented with additional nutrients. Bacterial growth parameters were assessed using OD<sub>620</sub> measurement. It was shown that whey supplemented with yeast extract supported the best growth, allowing a μ<sub>max</sub> = 0.63 h<sup>−1</sup>.
Ana Laura Reyes-Reyes, Francisco Valero, Georgina Sandoval
Background: GDSL-like esterase/lipase proteins (GELPs) are enzymes that possess unique characteristics, they contain four invariable catalytic residues. Advances in the study of these proteins are interesting. The cloning and functional expression of a papaya esterase have not been reported. Therefore, in this work we evaluated the heterologous production of Carica papaya esterase CpEST in the yeast Komogataella phaffii (Pichia pastoris). Results: The cloning and expression of the protein was performed under the PAOX1 promoter, and productions of up to 43 AU/mL were achieved using residual glycerol from biodiesel in the batch phase and methanol for the induction phase. Enzyme activity assays determined that CpEST has a high preference for short-chain substrates (p-NP C4 and p-NP C8), and optimal activity conditions were observed at 30°C and pH 10. The enzyme showed the highest stability to acetone, ethanol and tert-butanol solvents, retaining approximately 55% of its initial enzymatic activity after 1 h of exposure. Conclusions: Cloning and functional expression of papaya CpEST esterase was achieved. During fermentation, the yeasts used as a carbon source residual glycerol from biodiesel production. Based on the results obtained from the characterization of the esterase, it was found that it has a high potential for use in the bioenergy and detergent industry.How to cite: Reyes-Reyes AL, Valero F, Sandoval G. Cloning, protein expression and biochemical characterization of Carica papaya esterase. Electron J Biotechnol 2022;61. https://doi.org/10.1016/j.ejbt.2022.11.004.
Muhammad A. Butt, Nikolay L. Kazanskiy, Svetlana N. Khonina
A Bragg grating (BG) is a one-dimensional optical device that may reflect a specific wavelength of light while transmitting all others. It is created by the periodic fluctuation of the refractive index in the waveguide (WG). The reflectivity of a BG is specified by the index modulation profile. A Bragg grating is a flexible optical filter that has found broad use in several scientific and industrial domains due to its straightforward construction and distinctive filtering capacity. WG BGs are also widely utilized in sensing applications due to their easy integration and high sensitivity. Sensors that utilize optical signals for sensing have several benefits over conventional sensors that use electric signals to achieve detection, including being lighter, having a strong ability to resist electromagnetic interference, consuming less power, operating over a wider frequency range, performing consistently, operating at a high speed, and experiencing less loss and crosstalk. WG BGs are simple to include in chips and are compatible with complementary metal-oxide-semiconductor (CMOS) manufacturing processes. In this review, WG BG structures based on three major optical platforms including semiconductors, polymers, and plasmonics are discussed for filtering and sensing applications. Based on the desired application and available fabrication facilities, the optical platform is selected, which mainly regulates the device performance and footprint.
Markéta Macho, Subhasish Saha, Grzegorz Konert
et al.
ABSTRACT Resistance development and exhaustion of the arsenal of existing antibacterial agents urgently require an alternative approach toward drug discovery. Herein, we report the screening of Medicines for Malaria Venture (MMV) Pandemic Response Box (PRB) through a cascade developed to streamline the potential compounds with antivirulent properties to combat an opportunistic pathogen, Pseudomonas aeruginosa. To find an agent suppressing the production of P. aeruginosa virulence factors, we assessed the potential of the compounds in PRB with quorum sensing inhibitory activity. Our approach led us to identify four compounds with significant inhibition of extracellular virulence factor production and biofilm formation. This provides an opportunity to expand and redirect the application of these data sets toward the development of a drug with unexplored target-based activity. IMPORTANCE The rise of drug-resistant pathogens as well as overuse and misuse of antibiotics threatens modern medicine as the number of effective antimicrobial drugs steadily decreases. Given the nature of antimicrobial resistance development under intense selective pressure such as the one posed by pathogen-eliminating antibiotics, new treatment options which could slow down the emergence of resistance are urgently needed. Antivirulence therapy aims at suppressing a pathogen’s ability to cause disease rather than eliminating it, generating significantly lower selective pressure. Quorum sensing inhibitors are thought to be able to downregulate the production of virulence factors, allowing for smaller amounts of antimicrobials to be used and thus preventing the emergence of resistance. The PRB constitutes an unprecedented opportunity to repurpose new as well as known compounds with cytotoxicity and in vitro absorption, distribution, metabolism and excretion (ADME) profile available, thus shortening the time between compound discovery and medicinal use.
Acute myeloid leukemia (AML) is an infrequent disease, and it is associated with high morbidity and mortality. It harbors a unique configuration of cytogenetic abnormalities and molecular mutations that can be detected using microscopic and molecular methods respectively. These genetic tests are core elements of diagnosis and prognostication in high-income countries. They are routinely incorporated in clinical decision making, allowing for the individualization of therapy. However, these tests are largely inaccessible to most patients in Kenya and therefore no data has been reported on this group of patients. The main purpose of this study is to describe the cytogenetic and molecular abnormalities of acute myeloid leukemia patients seen at the hemato-oncology unit of Kenyatta National Hospital. A cross-sectional descriptive study was carried out over a 3-month period on ten patients with a diagnosis of AML. Social demographics and clinical data were collected through a study proforma. A peripheral blood sample was collected for conventional metaphase G-banding technique and next generation sequencing. Particularly, targeted DNA sequencing (Illumina myeloid panel) and whole exome sequencing (WES) were performed. Cytogenetic analysis failed in 10/10 cases. Targeted sequencing was successfully obtained in 8 cases, whereas WES in 7. Cytogenetic studies yielded no results. There were 20 mutations detected across 10 commonly mutated genes. All patients had at least one clinically relevant mutation. Based on ELN criteria, NGS identified three patients with high-risk mutations, affecting TP53 (n = 2) and RUNX1 (n = 1). One patient was classified as favorable (PML-RARA) while 4 were standard risk. However, WT1 mutations associated with unfavorable prognosis were recorded in additional 2 cases. WES showed concordant results with targeted sequencing while unveiling more mutations that warrant further attention. In conclusion, we provide the first molecular profiling study of AML patients in Kenya including application of advanced next generation sequencing technologies, highlighting current limitations of AML diagnostics and treatment while confirming the relevance of NGS in AML characterization.
Humic substances (HSs) occupy 80% of organic matter in soil and have been widely applied for soil remediation agents, potential battery materials, and adsorbents. Since the HS extraction rate is very low by microbial degradation in nature, artificial humification processes such as aerobic composting (AC) and hydrothermal treatment (HT) have attracted a great deal of attention as the most important strategies in HS production. This article aims to provide a state-of-the-art review on the development of conversion of biomass waste into HSs based on AC and HT for the first time in terms of mechanisms, characteristics of HSs’ molecular structure, and influencing factors. In addition, some differences based on the aforementioned information between AC and HT are reviewed and discussed in the conversion of biomass waste into HSs in a pioneering way. For biomass waste conversion, a feasible strategy on effective humification processes by combining AC with HT is proposed.
Yu-Tzu Chang, Tsai-Chieh Ling, Ya-Yun Cheng
et al.
Background: Non-dialysis-dependent chronic kidney disease (CKD-ND) patients are recommended to receive a one-dose influenza vaccination annually. However, studies investigating vaccine efficacy in the CKD-ND population are still lacking. In this study, we aimed to evaluate vaccine efficacy between the one-dose and two-dose regimen and among patients with different stages of CKD throughout a 20-week follow-up period. Methods: We conducted a single-center, non-randomized, open-label, controlled trial among patients with all stages of CKD-ND. Subjects were classified as unvaccinated, one-dose, and two-dose groups (4 weeks apart) after enrollment. Serial changes in immunological parameters (0, 4, 8, and 20 weeks after enrollment), including seroprotection, geometric mean titer (GMT), GMT fold-increase, seroconversion, and seroresponse, were applied to evaluate vaccine efficacy. Results: There were 43, 84, and 71 patients in the unvaccinated, one-dose, and two-dose vaccination groups, respectively. At 4–8 weeks after vaccination, seroprotection rates in the one- and two-dose group for H1N1, H3N2, and B ranged from 82.6–95.8%, 97.4–100%, and 73.9–100%, respectively. The concomitant seroconversion and GMT fold-increases nearly met the suggested criteria for vaccine efficacy for the elderly population. Although the seroprotection rates for all of the groups were adequate, the seroconversion and GMT fold-increase at 20 weeks after vaccination did not meet the criteria for vaccine efficacy. The two-dose regimen had a higher probability of achieving seroprotection for B strains (Odds ratio: 3.5, 95% confidence interval (1.30–9.40)). No significant differences in vaccine efficacy were found between early (stage 1–3) and late (stage 4–5) stage CKD. Conclusions: The standard one-dose vaccination can elicit sufficient protective antibodies. The two-dose regimen induced a better immune response when the baseline serum antibody titer was low. Monitoring change in antibody titers for a longer duration is warranted to further determine the current vaccine strategy in CKD-ND population.
Leszek Potocki, Bernadetta Oklejewicz, Ewelina Kuna
et al.
As microalgae are producers of proteins, lipids, polysaccharides, pigments, vitamins and unique secondary metabolites, microalgal biotechnology has gained attention in recent decades. Microalgae can be used for biomass production and to obtain biotechnologically important products. Here, we present the application of a method of producing a natural, biologically active composite obtained from unicellular microalgae of the genus <i>Planktochlorella</i> sp. as a modulator of the growth of microorganisms that can be used in the cosmetics and pharmaceutical industries by exploiting the phenomenon of photo-reprogramming of metabolism. The combination of red and blue light allows the collection of biomass with unique biochemical profiles, especially fatty acid composition (Patent Application P.429620). The ethanolic and water extracts of algae biomass inhibited the growth of a number of pathogenic bacteria, namely <i>Enterococcus faecalis</i>, <i>Staphylococcus aureus</i> PCM 458, <i>Streptococcus pyogenes</i> PCM 2318, <i>Pseudomonas aeruginosa</i>, <i>Escherichia coli</i> PCM 2209 and <i>Candida albicans</i> ATCC 14053. The algal biocomposite obtained according to our procedure can be used also as a prebiotic supplement. The presented technology may allow the limitation of the use of antibiotics and environmentally harmful chemicals commonly used in preparations against <i>Enterococcus faecalis</i>, <i>Staphylococcus aureus</i>, <i>Streptococcus pyogenes</i>, <i>Pseudomonas aeruginosa</i>, <i>Escherichia coli</i> or <i>Candida</i> spp.
Summary: The RET proto-oncogene encodes receptor tyrosine kinase, expressed primarily in tissues of neural crest origin. De-regulation of RET signaling is implicated in several human cancers. Recent phosphatome interactome analysis identified PTPRA interacting with the neurotrophic factor (GDNF)-dependent RET-Ras-MAPK signaling-axis. Here, by identifying comprehensive interactomes of PTPRA and RET, we reveal their close physical and functional association. The PTPRA directly interacts with RET, and using the phosphoproteomic approach, we identify RET as a direct dephosphorylation substrate of PTPRA both in vivo and in vitro. The protein phosphatase domain-1 is indispensable for the PTPRA inhibitory role on RET activity and downstream Ras-MAPK signaling, whereas domain-2 has only minor effect. Furthermore, PTPRA also regulates the RET oncogenic mutant variant MEN2A activity and invasion capacity, whereas the MEN2B is insensitive to PTPRA. In sum, we discern PTPRA as a novel regulator of RET signaling in both health and cancer. : Biological Sciences; Molecular Biology; Cancer Subject Areas: Biological Sciences, Molecular Biology, Cancer
Biogenic silver nanoparticles (Ag NPs) have supple platforms designed for biomedical and therapeutic intervention. Utilization of Ag NPs are preferred in the field of biomedicines and material science research because of their antioxidant, antimicrobial, and anticancerous activity along with their eco-friendly, biocompatible, and cost-effective nature. Here we present a novel fungus Piriformospora indica as an excellent source for obtaining facile and reliable Ag NPs with a high degree of consistent morphology. We demonstrated their cytotoxic property, coupled with their intrinsic characteristic that make these biogenic nanoparticles suitable for the anticancerous activity. In vitro cytotoxicity of biologically synthesized Ag NPs (BSNPs) and chemically synthesized Ag NPs (SNPs) was screened on various cancer cell lines, such as Human breast adenocarcinoma (MCF-7), Human cervical carcinoma (HeLa), Human liver hepatocellular carcinoma (HepG2) cell lines and embryonic kidney cell line (HEK-293) as normal cell lines. The antiproliferative outcome revealed that the BSNPs exhibited significant cytotoxic activity against MCF-7 followed by HeLa and HepG2 cell lines as compared to SNPs. The blend of cytotoxic properties, together with green and cost-effective characteristics make up these biogenic nanoparticles for their potential applications in cancer nanomedicine and fabrication coating of ambulatory and non-ambulatory medical devices.
Andreas Leimbach, Anja Poehlein, John Vollmers
et al.
Abstract Background Escherichia coli bovine mastitis is a disease of significant economic importance in the dairy industry. Molecular characterization of mastitis-associated E. coli (MAEC) did not result in the identification of common traits. Nevertheless, a mammary pathogenic E. coli (MPEC) pathotype has been proposed suggesting virulence traits that differentiate MAEC from commensal E. coli. The present study was designed to investigate the MPEC pathotype hypothesis by comparing the genomes of MAEC and commensal bovine E. coli. Results We sequenced the genomes of eight E. coli isolated from bovine mastitis cases and six fecal commensal isolates from udder-healthy cows. We analyzed the phylogenetic history of bovine E. coli genomes by supplementing this strain panel with eleven bovine-associated E. coli from public databases. The majority of the isolates originate from phylogroups A and B1, but neither MAEC nor commensal strains could be unambiguously distinguished by phylogenetic lineage. The gene content of both MAEC and commensal strains is highly diverse and dominated by their phylogenetic background. Although individual strains carry some typical E. coli virulence-associated genes, no traits important for pathogenicity could be specifically attributed to MAEC. Instead, both commensal strains and MAEC have very few gene families enriched in either pathotype. Only the aerobactin siderophore gene cluster was enriched in commensal E. coli within our strain panel. Conclusions This is the first characterization of a phylogenetically diverse strain panel including several MAEC and commensal isolates. With our comparative genomics approach we could not confirm previous studies that argue for a positive selection of specific traits enabling MAEC to elicit bovine mastitis. Instead, MAEC are facultative and opportunistic pathogens recruited from the highly diverse bovine gastrointestinal microbiota. Virulence-associated genes implicated in mastitis are a by-product of commensalism with the primary function to enhance fitness in the bovine gastrointestinal tract. Therefore, we put the definition of the MPEC pathotype into question and suggest to designate corresponding isolates as MAEC.
This work was carried out to study the effect of Diammonium Phosphate (DAP) fertilizer during spring 2014 to produce the volatile oil from sage plant. Four levels of DAP (21%P, 18%N) (0, 100, 200 and 300 Kg/ha) were used on sage plants. The experiment was designed according to complete randomized design (CRD). The measurements of plant heights, Fresh and dry weights, surface area of the aerial leaves and active compound concentration were recorded as control before flowering. Results revealed significant differences (p ≤0.05) in Plants height, fresh and dry weights and surface area between treatments. In the control group measures were (18.00cm, 100.22 g/pot, 18.55 g/pot and 5.22 cm2) respectively. The highest values were reported with 300 kg/ha treatment (26.50 cm, 118.25, 21.86 g/pot and 7.21 cm2) respectively. Volatile concentration was measured using Clevenger with steam distillation apparatus. Diammonium Phosphate addition levels showed different effect on Volatile oil content in aerial part (Shoots). The lowest was (1.68%) for 300 kg/ha, and the highest was (3.07%) in 100 Kg/ha
Christian A. Hernández, Norma Sandoval, Julieta Mallerman
et al.
Background: Ethanol has been pointed out as a laccase inducer. However, there are controversial reports about its efficiency with some fungi. In this study, we hypothesized that ethanol laccase induced in Pycnoporus sanguineus depends on nitrogen nutriment conditions. To prove this, we assessed laccase production in submerged cultures of P. sanguineus, with different nitrogen concentrations and with, or without ethanol added in a factorial designed experiment.
Results: In order to analyze the effects of factors on the response variables, a factorial ANOVA, and response-surface models were performed. It was found that the nitrogen source was the main factor that affected laccase production in P. sanguineus. The treatments with yeast extract (2 g/L) and ethanol (3 g/L) induced the highest laccase activity (31.01 ± 4.9 U/L), while the treatments with urea reached the lowest activity (less than 1.6 U/L). Ethanol had positive and synergic effects on laccase production, in accordance with the surface response model, as long as simple nitrogen sources (urea) were not available.
Conclusions: We suggest that laccase in P. sanguineus is regulated by a catabolic nitrogen repression mechanism; laccase activity is strongly inhibited by urea used as nitrogen source and it decreases when the amount of urea increases; contrarily, a synergic positive effect was observed between yeast extract and ethanol on laccase production.