Culturable and unculturable potential heterotrophic microbiological threats to the oldest pyramids of the Memphis necropolis, Egypt.

A large percentage of the world's tangible cultural heritage is made from stone; thus, it deteriorates due to physical, chemical, and/or biological factors. The current study explored the microbial community inhabiting two prehistoric sites with high cultural value in the Memphis necropolis of Egypt (Djoser and Lahun Pyramids) using amplicon-based metabarcoding and culture-dependent isolation methods. Samples were examined by epifluorescent microscopy for biological signs before environmental DNA extraction and in vitro cultivation. The metabarcoding analysis identified 644 bacterial species (452 genera) using the 16S rRNA and 204 fungal species (146 genera) using ITS. In comparison with the isolation approach, an additional 28 bacterial species (13 genera) and 34 fungal species (20 genera) were identified. A total of 19 bacterial and 16 fungal species were exclusively culture-dependent, while 92 bacterial and 122 fungal species were culture-independent. The most abundant stone-inhabiting bacteria in the current study were Blastococcus aggregatus, Blastococcus saxobsidens, and Blastococcus sp., among others. The most abundant rock-inhabiting fungi were Knufia karalitana and Pseudotaeniolina globosa, besides abundant unknown Sporormiaceae species. Based on previous reports, microorganisms associated with biodeterioration were detected on color-altered sites at both pyramids. These microorganisms are potentially dangerous as physical and chemical deterioration factors and require proper conservation plans from a microbiological perspective.

A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms.

Reference-free reduced representation bisulfite sequencing uses enzymatic digestion for reducing genome complexity and allows detection of markers to study DNA methylation of a high number of individuals in natural populations of non-model organisms. Current methods like epiGBS enquire the use of a higher number of methylated DNA oligos with a significant cost (especially for small labs and first pilot studies). In this paper, we present a modification of this epiGBS protocol that requires the use of only one hemimethylated P2 (common) adapter, which is combined with unmethylated barcoded adapters. The unmethylated cytosines of one chain of the barcoded adapter are replaced by methylated cytosines using nick translation with methylated cytosines in dNTP solution. The basic version of our technique uses only one restriction enzyme, and as a result, genomic fragments are integrated into two orientations with respect to the adapter sequences. Comparing the sequences of two chain orientations makes it possible to reconstruct the original sequence before bisulfite treatment with the help of standard software and newly developed software written in C and described here. We provide a proof of concept via data obtained from almond (Prunus dulcis). Example data and a detailed description of the complete software pipeline starting from the raw reads up until the final differentially methylated cytosines are given in Supplementary Material making this technique accessible to non-expert computer users. The adapter design showed in this paper should allow the use of a two restriction enzyme approach with minor changes in software parameters.

In vitro lead tolerance and accumulation in three Chrysanthemum cultivars for phytoremediation purposes with ornamental plants.

The use of ornamental plants for the phytoremediation of potentially toxic elements in polluted soils is an interesting task. It makes possible to combine environmental restoration, re-use of land, and the production of goods and services of economic interest. In this work, in vitro experiments using three cultivars of Chrysanthemum (Asteraceae) were carried out with 0, 300, 600, 900, and 1500 mg/kg of lead concentrations for a period of 12 weeks. The objective was to obtain data about their lead tolerance and bioaccumulation capacity in order to know their potential as phytoremediators in a densely populated Caribbean area of the Dominican Republic with a high concentration of lead in soils. The variations in biomass, root growth as well as accumulation of this element in the plants were measured. The results suggest that the three cultivars have a good potential for phytoextraction at moderate pollution levels, as they showed a good bioaccumulation of lead, which had mild effects on their biomass production and root elongation. Additional studies should be carried out to assess their effectiveness as phytoextractors under field conditions, as well as other alternative uses that could generate esthetic, environmental, and/or economic benefits for tropical areas contaminated by Pb.

Anthocyanins of Blue Corn and Tortilla Arrest Cell Cycle and Induce Apoptosis on Breast and Prostate Cancer Cells.

Background: Breast and prostate cancer are frequently diagnosed neoplasias in women and men around the world. The signaling of the androgen receptor (AR) influences the development of both tumors. Since therapies focused to block the receptor's activity have not been fully effective, and have shown side effects, therapies based on natural compounds are promissory complementary alternatives in its treatment. Objective: The aim of this study was to determine the effect of anthocyanins from blue corn in cancer cell lines. Methods: We analyzed the antiproliferative effect of anthocyanins from raw and alkali-processed (tortillas) Mixteco blue corn in breast and prostate cancer cell lines MDA-MB-453 (subtype: triple negative) and LNCaP using methyltiazlyl-tetrazolium (MTT) and flow cytometry (FCM). The combination of anthocyanins and 2-amino-N-quinolin-8-yl-benzenesulfonamide (QBS) or nocodazole also were evaluated. The anthocyanins were isolated trough column chromatography (XAD-7).Results: Our results demonstrated that anthocyanin specially the ones obtained from tortillas, decreased cell viability and arrested cell cycle in G1 phase inducing apoptosis. Cytometry analysis shows an increased effect on apoptosis of MDA-MB-453 and LNCaP cells when tortilla anthocyanins and QBS were combined. Conclusions: This is the first report that suggest that anthocyanins from blue corn have an effect in cell cycle and viability so they could serve as adjuvants for breast and prostate cancer therapies and may prompt to deepen investigations to decipher its molecular properties. AbbreviationsARAndrogen ReceptorCIDIIRInterdisciplinary Center for Research on Integral Regional DevelopmentDHT5α-DihydrotestosteroneEREstrogen ReceptorPRProgesterone ReceptorQBSAmino-N-quinolin-8-yl-benzenesulfonamide.

DNA Methylation Analysis of Dormancy Release in Almond (Prunus dulcis) Flower Buds Using Epi-Genotyping by Sequencing.

DNA methylation and histone post-translational modifications have been described as epigenetic regulation mechanisms involved in developmental transitions in plants, including seasonal changes in fruit trees. In species like almond (Prunus dulcis (Mill.) D.A: Webb), prolonged exposure to cold temperatures is required for dormancy release and flowering. Aiming to identify genomic regions with differential methylation states in response to chill accumulation, we carried out Illumina reduced-representation genome sequencing on bisulfite-treated DNA from floral buds. To do this, we analyzed almond genotypes with different chilling requirements and flowering times both before and after dormancy release for two consecutive years. The study was performed using epi-Genotyping by Sequencing (epi-GBS). A total of 7317 fragments were sequenced and the samples compared. Out of these fragments, 677 were identified as differentially methylated between the almond genotypes. Mapping these fragments using the Prunus persica (L.) Batsch v.2 genome as reference provided information about coding regions linked to early and late flowering methylation markers. Additionally, the methylation state of ten gene-coding sequences was found to be linked to the dormancy release process.

Peripatric speciation associated with genome expansion and female-biased sex ratios in the moss genus Ceratodon.

PREMISE OF THE STUDY: A period of allopatry is widely believed to be essential for the evolution of reproductive isolation. However, strict allopatry may be difficult to achieve in some cosmopolitan, spore-dispersed groups, like mosses. We examined the genetic and genome size diversity in Mediterranean populations of the moss Ceratodon purpureus s.l. to evaluate the role of allopatry and ploidy change in population divergence. METHODS: We sampled populations of the genus Ceratodon from mountainous areas and lowlands of the Mediterranean region, and from Western and Central Europe. We performed phylogenetic and coalescent analyses on sequences from five nuclear introns and a chloroplast locus to reconstruct their evolutionary history. We also estimated genome size using flow cytometry (employing propidium iodide) and determined the sex of samples using a sex-linked PCR marker. KEY RESULTS: Two well-differentiated clades were resolved, discriminating two homogeneous groups: the widespread C. purpureus and a local group mostly restricted to the mountains in Southern Spain. The latter also possessed a genome size 25% larger than the widespread C. purpureus, and the samples of this group consist entirely of females. We also found hybrids, and some of them had a genome size equivalent to the sum of the C. purpureus and Spanish genome, suggesting that they arose by allopolyploidy. CONCLUSIONS: These data suggest that a new species of Ceratodon arose via peripatric speciation, potentially involving a genome size change and a strong female-biased sex ratio. The new species has hybridized in the past with C. purpureus.

Antioxidant and antiproliferative activity of blue corn and tortilla from native maize.

BACKGROUND: Blue corn is a cereal rich in phenolic compounds used to make blue tortillas. Tortillas are an important part of the Mexican diet. Blue corn and tortilla represent an important source of the natural antioxidants anthocyanins. However, studies on their biological activity on cancer cell lines are limited. The goal of this study was to evaluate the antioxidant and antiproliferative activity of blue corn and tortilla on different cancer cell lines. METHODS: Total polyphenol content, monomeric anthocyanins, and antioxidant activity by the DPPH and TBARS methods of blue corn and tortilla were determined. The anthocyanin profile of tortilla was obtained by means of HPLC-ESI-MS. The antiproliferative activity of blue corn and tortilla extract on HepG2, H-460, Hela, MCF-7 and PC-3 was evaluated by the MTT assay. RESULTS: Blue corn had higher content of total polyphenols and monomeric anthocyanins as well as lower percentage of polymeric color than tortilla; however, both showed similar antioxidant activity by DPPH. In addition, although a higher degradation of anthocyanins was observed on tortilla extract, both extracts inhibited lipid peroxidation (IC50) at a similar concentration. The anthocyanin profile showed 28 compounds which are primarily derived from cyanidin, including acylated anthocyanins and proanthocyanidins. Blue corn and tortilla extracts showed antiproliferative effects against HepG2, H-460, MCF-7 and PC-3 cells at 1000 µg/mL, however Hela cells were more sensitive at this concentration. CONCLUSION: This is the first report to demonstrate anticancer properties in vitro of tortilla derived from blue corn, suggesting that this product has beneficial health effects. In addition, blue corn could be a potential source of nutraceuticals with anticancer activity.

Elevational patterns of genetic variation in the cosmopolitan moss Bryum argenteum (Bryaceae).

PREMISE OF THE STUDY: The Baas Becking tenet posits that 'everything is everywhere, but the environment selects' to explain cosmopolitan distributions in highly vagile taxa. Bryophyte species show wider distributions than vascular plants and include examples of truly cosmopolitan ranges, which have been interpreted as a result of high dispersal capacities and ecological plasticity. In the current study, we documented patterns of genetic structure and diversity in the cosmopolitan moss Bryum argenteum along an elevational gradient to determine if genetic diversity and structure is homogenized by intense migrations in the lack of ecological differentiation. • METHODS: 60 specimens were collected in the Sierra Nevada Mountains (Spain) between 100 and 2870 m and sequenced for ITS and rps4. Comparative analyses, genetic diversity estimators, and Mantel's tests were employed to determine the relationship between genetic variation, elevation, and geographic distance and to look for signs of demographic shifts. • KEY RESULTS: Genetic diversity peaked above 1900 m and no signs of demographic shifts were detected at any elevation. There was a strong phylogenetic component in elevational variation. Genetic variation was significantly correlated with elevation, but not with geographic distance. • CONCLUSIONS: The results point to the long-term persistence of Bryum argenteum in a range that was glaciated during the Late Pleistocene. Evidence for an environmentally driven pattern of genetic differentiation suggests adaptive divergence. This supports the Baas Becking tenet and indicates that ecological specialization might play a key role in explaining patterns of genetic structure in cosmopolitan mosses.

High hydrostatic pressure induces synthesis of heat-shock proteins and trehalose-6-phosphate synthase in Anastrepha ludens larvae.

The Mexican fruit fly (Anastrepha ludens) is responsible for losses of up to 25% of crops such as mango and citrus fruits in Central America and México. The larval life cycle of A. ludens comprises three stages with a duration ranging from 3 to 8 days. Because of the damage caused by A. ludens, several methods of control have been studied and implemented. High hydrostatic pressures (HHP) are currently applied to foods and it is now proposed to be employed to inactivate eggs and larvae of A. ludens. Originally HHP was designed to inactivate microorganisms, since it exerts marked effects on cell morphology, and can affect enzymatic reactions and genetic mechanisms of microbial cells, with no major changes altering the sensory or nutritional quality of the foodstuff. In this study, A. ludens in two larval stages (5- and 8-day-old) were subjected to HHP treatments. The biochemical response of the larvae of A. ludens was dependent on their stage of development. The third larval stage (L3) developed a better protection mechanism based on the synthesis of stress proteins or heat-shock proteins (HSPs) and the enzyme trehalose-6-phosphate synthase, which are linked and possibly act together to achieve greater survivability to stress caused by hydrostatic pressure.

Reclassification of Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov.

Although Anoxybacillus and Geobacillus, two genera of thermophilic bacteria close to the genus Bacillus, have only been described recently, the number of species in these genera has increased rapidly. Four thermophilic, lipolytic strains (DR01, DR02, DR03 and DR04) isolated from a hot spring in Veracruz (Mexico), which could not be identified phenotypically, were subjected to 16S rRNA gene sequence analysis. Three strains were identified as belonging to the genus Anoxybacillus, but strain DR03 was identified as Geobacillus pallidus. This result led us to perform a phylogenetic analysis of the genera Anoxybacillus and Geobacillus based on 16S rRNA gene sequences from all the type strains of these genera. Phylogenetic trees showed three major clusters, Anoxybacillus-Geobacillus tepidamans, Geobacillus sensu stricto and Geobacillus pallidus, while the 16S rRNA gene sequences of G. pallidus (DR03 and the type strain) showed low similarity to sequences of Anoxybacillus (92.5-95.1 %) and Geobacillus (92.8-94.5 %) species, as well as to Bacillus subtilis (92.2-92.4 %). In addition, G. pallidus could be differentiated from Anoxybacillus and Geobacillus on the basis of DNA G+C content and fatty acid and polar lipid profiles. From these results, it is proposed that Geobacillus pallidus should be classified in a novel genus, for which we propose the name Aeribacillus, as Aeribacillus pallidus gen. nov., comb. nov. The type strain of Aeribacillus pallidus is H12(T) (=ATCC 51176(T) =DSM 3670(T) =LMG 19006(T)).

Induction of Cd36 expression elicited by fish oil PUFA in spontaneously hypertensive rats.

Cd36 is an integral membrane glycoprotein expressed on the surface of cells active in fatty acid metabolism (adipocytes, muscle cells, platelets, monocytes, heart and intestine cells). This protein plays diverse functions including uptake of long-chain fatty acids and oxidized low-density lipoproteins. A recent report demonstrates that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridemia in spontaneously hypertensive rats (SHRs). Cd36 is a tightly regulated protein whose expression is modulated through peroxisome proliferator-activated receptor (PPAR) transcription factors, by conditions that alter lipid metabolism such as diabetes mellitus and high-fat feeding. The purpose of this study was to evaluate the effect of dietary fish oil, rich in n-3 polyunsaturated fatty acids (PUFAs), on metabolic parameters and on the expression levels of Cd36 in adipose tissue in the SHR. Spontaneously hypertensive rats showed lower Cd36 mRNA levels when compared to Kyoto-Wistar (KW) rats (control). After 6 weeks of fish oil (FO) administration, this group of SHRs (FO-SHR) presented increased levels of Cd36 mRNA, concomitantly with decreased insulin, free fatty acids (FFAs), triglycerides, cholesterol, LDL, HDL, total lipids and blood pressure, in comparison to control rats that received a corn-canola oil diet. The study confirmed the beneficial effects of fish oil administration on the metabolic syndrome, suggesting that the induction of Cd36 expression could be one of the molecular mechanisms elicited by fish oil PUFAs.

Effects of fish oil on hypertension, plasma lipids, and tumor necrosis factor-alpha in rats with sucrose-induced metabolic syndrome.

Dietary fish oil rich in (n-3) fatty acids plays an important role in reducing abnormalities associated with the metabolic syndrome and mortality from coronary heart disease. We investigated the effects of dietary fish oil on the metabolic syndrome in a high-sucrose-fed rat model. The model was achieved by the administration of 30% sucrose in drinking water in male Wistar rats during 21 weeks. After the metabolic syndrome rat model was established, fish oil was administered during 6 weeks. The metabolic syndrome rats showed significant increases in body weight, systolic blood pressure, serum insulin, total lipids, triacylglycerols, cholesterol, free fatty acids, LDL, total proteins, albumin, and serum tumor necrosis factor-alpha (TNF-alpha). They also presented abdominal and epididymal fat accumulation and fatty liver. After fish oil diet administration, metabolic syndrome rats had a significant reduction in blood pressure, serum insulin, triacylglycerols, cholesterol, free fatty acids, and total lipids, but no change was observed in TNF-alpha concentration or fat accumulation. In conclusion, fish oil reversed the alterations on metabolic parameters and blood pressure exerted by sucrose administration, although it had no effect on TNF-alpha production and adiposity. This confirms the theory that the molecular etiology of the metabolic syndrome is multifactorial, as is the effect of n-3 polyunsaturated fatty acids (PUFAs) upon it, having complex and multifaceted actions.

Intercontinental Mediterranean disjunct mosses: morphological and molecular patterns.

This study focused on three species that occur disjunctly between western North America and the Mediterranean region of southern Europe, northern Africa, and western Asia, forming the so-called Madrean-Tethyan distribution pattern. Quantitative morphological characters were measured in New and Old World plants to find any subtle phenotypic differentiation between the disjunct populations. Sequences from the nuclear ribosomal internal transcribed spacer region were obtained from the same populations to assess differentiation at the molecular level and to compare molecular diversity with patterns of morphological similarity among plants. Little or no morphological differentiation existed between New and Old World plants in any of the species, but internal transcribed spacer (ITS) sequences revealed some phylogeographic structure. Patterns of morphological similarity in all three species were incongruent with phylogeographic structure revealed by sequence data. New World populations were more variable than Old World populations at the molecular level in the three species. Despite some evidence for differentiation between disjunct plants, no plausible mutation rate would date the divergence at ≥20 million years ago (MYA), as implied by the Madrean-Tethyan hypothesis. Recent long-distance dispersal is a more likely explanation for intercontinental disjunctions in these species.