Novel spore-forming species exhibiting intrinsic resistance to third- and fourth-generation cephalosporins and description of Tigheibacillus jepli gen. nov., sp. nov.

A comprehensive microbial surveillance was conducted at NASA's Mars 2020 spacecraft assembly facility (SAF), where whole-genome sequencing (WGS) of 110 bacterial strains was performed. One isolate, designated 179-BFC-A-HST, exhibited less than 80% average nucleotide identity (ANI) to known species, suggesting a novel organism. This strain demonstrated high-level resistance [minimum inhibitory concentration (MIC) >256 mg/L] to third-generation cephalosporins, including ceftazidime, cefpodoxime, combination ceftazidime/avibactam, and the fourth-generation cephalosporin cefepime. The results of a comparative genomic analysis revealed that 179-BFC-A-HST is most closely related to Virgibacillus halophilus 5B73CT, sharing an ANI of 78.7% and a digital DNA-DNA hybridization (dDDH) value of 23.5%, while their 16S rRNA gene sequences shared 97.7% nucleotide identity. Based on these results and the recent recognition that the genus Virgibacillus is polyphyletic, strain 179-BFC-A-HST is proposed as a novel species of a novel genus, Tigheibacillus jepli gen. nov., sp. nov (type strain 179-BFC-A-HST = DSM 115946T = NRRL B-65666T), and its closest neighbor, V. halophilus, is proposed to be reassigned to this genus as Tigheibacillus halophilus comb. nov. (type strain 5B73CT = DSM 21623T = JCM 21758T = KCTC 13935T). It was also necessary to reclassify its second closest neighbor Virgibacillus soli, as a member of a novel genus Paracerasibacillus, reflecting its phylogenetic position relative to the genus Cerasibacillus, for which we propose Paracerasibacillus soli comb. nov. (type strain CC-YMP-6T = DSM 22952T = CCM 7714T). Within Amphibacillaceae (n = 64), P. soli exhibited 11 antibiotic resistance genes (ARG), while T. jepli encoded for 3, lacking any known ß-lactamases, suggesting resistance from variant penicillin-binding proteins, disrupting cephalosporin efficacy. P. soli was highly resistant to azithromycin (MIC >64 mg/L) yet susceptible to cephalosporins and penicillins. IMPORTANCE: The significance of this research extends to understanding microbial survival and adaptation in oligotrophic environments, such as those found in SAF. Whole-genome sequencing of several strains isolated from Mars 2020 mission assembly cleanroom facilities, including the discovery of the novel species Tigheibacillus jepli, highlights the resilience and antimicrobial resistance (AMR) in clinically relevant antibiotic classes of microbes in nutrient-scarce settings. The study also redefines the taxonomic classifications within the Amphibacillaceae family, aligning genetic identities with phylogenetic data. Investigating ARG and virulence factors (VF) across these strains illuminates the microbial capability for resistance under resource-limited conditions while emphasizing the role of human-associated VF in microbial survival, informing sterilization practices and microbial management in similar oligotrophic settings beyond spacecraft assembly cleanrooms such as pharmaceutical and medical industry cleanrooms.

Spore-based innovative paper-strip biosensor for the rapid detection of ß-lactam group in milk.

The study's goal was to develop a spore-based paper strip biosensor for detecting ß-lactam antibiotics in milk using the enzyme induction principle. A new spore-based paper strip biosensor has been developed after important operating parameters such as spore volume, substrate volume, exposure time and temperature, and incubation time and temperature were optimised. The limit of detection for various ß-lactam antibiotics, including amoxicillin, penicillin, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, cephalothin, cefalexin, cefoxitin, cefazolin, and cefuroxime, was determined in milk with detection sensitivity of 1 ppb, 2 ppb, 2 ppb, 10 ppb, 10 ppb, 10 ppb, 20 ppb, 10 ppb 1000 ppb, 10 ppb 300 ppb and 100 ppb, respectively. It was also tested with other contaminants such non-ß-lactam antibiotics, pesticides, aflatoxin, heavy metals, and other chemical contaminants, and no interference was found, indicating that the created biosensor had a low rate of false positive and negative results. In comparison to the AOAC-approved CHARM-ROSA ß-lactam strip test, which identified 7 raw milk and zero pasteurised milk samples positive for ß-lactam antibiotics, the sensor was further analysed and verified using 200 raw milk and 105 pasteurised milk samples. This indicates a perfect match between our biosensor and the AOAC-approved CHARM-ROSA ß-lactam strip test. The developed spore-based paper strip biosensors are expected to be useful in the rapid and cost-effective detection of ß-lactam antibiotic residues in milk samples at the dairy farm, reception dock, and production units, respectively.

Hybridization and spore dissection of native wine yeasts for improvement of ethanol resistance and osmotolerance.

Global warming has a significant impact on different viticultural parameters, including grape maturation. An increment of photosynthetic activity generates a rapid accumulation of sugars in the berry, followed by a dehydration process which leads to a higher concentration of soluble solids. This effect is exacerbated by current viticultural practices which favor the harvest of very mature grapes to obtain wines with sweet tannins. Considering the initial hyperosmotic stress conditions and the high ethanol concentration of the produced wine, fermentation of grape musts with high sugar content could be problematic for yeast starters. In the present study, we were able to obtain by classical hybridization and spore dissection methods one hybrid and one monosporic wine yeast strain with a combined ethanol and osmotolerant phenotype. The improved yeasts were tested in vinification trials with high sugar concentration and displayed excellent fermentation performance. Importantly, the obtained wines also showed good organoleptic properties during sensory analysis. Based on our results, we believed our improved hybrid and monosporic strains can be considered good alternatives to be used as yeast starters for fermentations with high sugar content.

In silico modelling and virtual screening for identification of inhibitors for spore wall protein-5 in Nosema bombycis.

Bombyx mori is an insect of economic importance in the production of silk. It often gets infected by Nosema bombycis, an intracellular parasite. The infection causes a fatal disease known as a Pebrine which affects the development of the worm. The infected larvae of silkworms are coated with brown spots and are unable to spin the silkworm thread. They lose appetite, become sluggish, opaque and ultimately die. The Spore Wall Protein 5 is an exospore protein in N. bombycis and interacts with the polar tube proteins PTP2 and PTP3, a part of the extrusion apparatus that facilitates infection of the host. SWP5 also plays an essential part in maintaining the structural integrity of the spore wall and could possibly regulate the route of the infection in N. bombycis. In the present study, the homology modelling of three protein structures SWP5, PTP2 and PTP3 were performed. The protein-protein interaction was studied and a complete complex of SWP5, PTP2 and PTP3 was generated to understand the discharge of the penetrating polar tube. Virtual screening and molecular dynamics simulation was performed and a potential lead-like molecule is identified.Communicated by Ramaswamy H. Sarma.

Development of a database and standardized approach for rpoB sequence-based subtyping and identification of aerobic spore-forming Bacillales.

Aerobic spore-forming Bacillales are a highly diverse and ubiquitous group that includes organisms that cause foodborne illnesses and food spoilage. Classical microbiological and biochemical identification of members of the order Bacillales represents a challenge due to the diversity of organisms in this group as well as the fact that the phenotypic-based taxonomic assignment of some named species in this group is not consistent with their phylogenomic characteristics. DNA-sequencing-based tools, on the other hand, can be fast and cost-effective, and can provide for a more reliable identification and characterization of Bacillales isolates. In comparison to 16S rDNA, rpoB was shown to better discriminate between Bacillales isolates and to allow for improved taxonomic assignment to the species level. However, the lack of a publicly accessible rpoB database, as well as the lack of standardized protocols for rpoB-based typing and strain identification, is a major challenge. Here, we report (i) the curation of a DNA sequence database for rpoB-based subtype classification of Bacillales isolates; (ii) the development of standardized protocols for generating rpoB sequence data, and a scheme for rpoB-based initial taxonomic identification of Bacillales isolates at the species level; and (iii) the integration of the database in a publicly accessible online platform that allows for the analysis of rpoB sequence data from uncharacterized Bacillales isolates. Specifically, we curated a database of DNA sequences for a 632-nt internal variable region within the rpoB gene from representative Bacillales reference type strains and a large number of isolates that we have previously isolated and characterized through multiple projects. As of May 21, 2021, the rpoB database contained more than 8350 rpoB sequences representing 1902 distinct rpoB allelic types that can be classified into 160 different genera. The database also includes 1129 rpoB sequences for representative Bacillales reference type strains as available on May 21, 2021 in the NCBI database. The rpoB database is integrated into the online Food Microbe Tracker platform (www.foodmicrobetracker.com) and can be queried using the integrated BLAST tool to initially subtype and taxonomically identify aerobic and facultative anaerobic spore-formers. While whole-genome sequencing is increasingly used in bacterial taxonomy, the rpoB sequence-based identification scheme described here provides a valuable tool as it allows for rapid and cost-effective initial isolate characterization, which can help to identify and characterize foodborne pathogens and food spoilage bacteria. In addition, the database and primers described here can also be adopted for metagenomics approaches that include rpoB as a target, improving discriminatory power and identification over what can be achieved using 16S rDNA as a target.

Efficacy and safety of spore-forming probiotics in the treatment of functional dyspepsia: a pilot randomised, double-blind, placebo-controlled trial.

BACKGROUND: Current treatments for functional dyspepsia have limited efficacy or present safety issues. We aimed to assess spore-forming probiotics in functional dyspepsia as monotherapy or add-on therapy to long-term treatment with proton-pump inhibitors. METHODS: In this single-centre, randomised, double-blind, placebo-controlled pilot trial that took place at University Hospitals Leuven (Leuven, Belgium), adult patients (≥18 years) with functional dyspepsia (as defined by Rome IV criteria, on proton-pump inhibitors or off proton-pump inhibitors) were randomly assigned (1:1) via computer-generated blocked lists, stratified by proton-pump inhibitor status, to receive 8 weeks of treatment with probiotics (Bacillus coagulans MY01 and Bacillus subtilis MY02, 2·5 × 109 colony-forming units per capsule) or placebo consumed twice per day, followed by an open-label extension phase of 8 weeks. Individuals with a history of abdominal surgery, diabetes, coeliac or inflammatory bowel disease, active psychiatric conditions, and use of immunosuppressant drugs, antibiotics, or probiotics in the past 3 months were excluded. All patients and on-site study personnel were masked to treatment allocation in the first 8 weeks. Symptoms, immune activation, and faecal microbiota were assessed and recorded. The primary endpoint was a decrease of at least 0·7 in the postprandial distress syndrome (PDS) score of the Leuven Postprandial Distress Scale in patients with a baseline PDS score of 1 or greater (at least mild symptoms), assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT04030780. FINDINGS: Between June 3, 2019, and March 11, 2020, of 93 individuals assessed for eligibility, we included 68 patients with functional dyspepsia (51 [75%] women, mean age 40·1 years [SD 14·4], 34 [50%] on proton-pump inhibitors). We randomly assigned 32 participants to probiotics and 36 to placebo. The proportion of clinical responders was higher with probiotics (12 [48%] of 25) than placebo (six [20%] of 30; relative risk 1·95 [95% CI 1·07-4·11]; p=0·028). The number of patients with adverse events was similar with probiotics (five [16%] of 32) and placebo (12 [33%] of 36). Two serious adverse events occurring during the open-label phase (appendicitis and syncope in two separate patients) were assessed as unlikely to be related to the study product. INTERPRETATION: In this exploratory study, B coagulans MY01 and B subtilis MY02 were efficacious and safe in the treatment of functional dyspepsia. Participants had potentially beneficial immune and microbial changes, which could provide insights into possible underlying mechanisms as future predictors or treatment targets. FUNDING: MY HEALTH.

In vitro spore germination and phytoremediation of Hg and Pb using gametophytes of Pityrogramma calomelanos.

Few pteridophytes have proven the capacity to accumulate and remediate heavy metals from contaminated soils. Pityrogramma calomelanos, a non-seasonal fast-growing, a cosmopolitan fern, is a good indicator of environmental conditions, was used in the present study. The life cycle of ferns alternates with haploid gametophyte and diploid sporophyte. The present study was undertaken to access the effect of mercury, in form of mercury (II) chloride [(HgCl2)] and lead as lead nitrate [Pb(NO3)2] in developmental studies using in vitro spore germination. Periodic recording of the germination, protonemal growth, rhizoid formation and differentiation of sex organs in different concentrations of heavy metals were conducted for a period of 6 weeks. It was found that the percentage of spore germination and the number of protonemal cells reduced significantly causing developmental defects in the presence of HgCl2 as compared to Pb(NO3)2 (p < 0.0001). A significant decrease in the number of archegonial count and chlorophyll content was observed in different concentrations of the heavy metals tested. Gametophytes of P. calomelanos recorded lead uptake of 646.51 ± 0.93 mg/kg in treatments of 25 ppm of lead and high mercury accumulation up to 1,885 ± 1.98 mg/kg at 10 ppm of mercury, indicating successful uptake of heavy metals. Novelty statement: Pityrogramma calomelanos is gaining interest amongst pteridologists upon proving its exclusive capacity of phytoremediation. It is superior in comparison to the most popular, patented fern Pteris vittata. Our study demonstrates the effective use of the promising fern in its simplest, nonvascular form of gametophytes as a phytoremediation agent in controlled conditions.

Methylobacterium symbioticum sp. nov., a new species isolated from spores of Glomus iranicum var. tenuihypharum.

Strain SB0023/3 T, isolated from spores of the arbuscular mycorrhizal fungus Glomus iranicum var. tenuihypharum, was analysed to determine whether it represents a new species. It was studied for its applicability in the field of agriculture to reduce the input of nitrogen fertilizers. Comparative analysis of the 16S rRNA gene sequence shows the strain to be affiliated to the genus Methylobacterium, the closest similarities (98.7%) being shared with Methylobacterium dankookense. Further phylogenomic analysis through Up-to-date Bacterial Core Gene (UBCG) confirmed Methylobacterium dankookense as its closest relative. Average Nucleotide Identity (ANI) and in silico DNA-DNA hybridization (DDH) were lower than 92% and 44%, respectively, of the values shown by its phylogenetic relatives. Its genome had an approximate length of 6.05 Mb and the G + C content of the genome was 70.1 mol%. The main cellular fatty acid was Summed Feature 8 (C18:1ω7c and/or C18:1ω6c). It is a Gram-staining-negative, pink-pigmented, strictly aerobic and facultative methylotroph; it grows at 28 ºC and can grow at up to 3% salinity in the presence of sodium chloride. All the data collected support the naming of a novel species to accommodate the strain SB0023/3 T, for which the name Methylobacterium symbioticum sp. nov. is proposed. The type strain is SB0023/3 T (=CECT 9862 T =PYCC 8351 T).

Species-Specific Biodegradation of Sporopollenin-Based Microcapsules.

Sporoderms, the outer layers of plant spores and pollen grains, are some of the most robust biomaterials in nature. In order to evaluate the potential of sporoderms in biomedical applications, we studied the biodegradation in simulated gastrointestinal fluid of sporoderm microcapsules (SDMCs) derived from four different plant species: lycopodium (Lycopodium clavatum L.), camellia (Camellia sinensis L.), cattail (Typha angustifolia L.), and dandelion (Taraxacum officinale L.). Dynamic image particle analysis (DIPA) and field-emission scanning electron microscopy (FE-SEM) were used to investigate the morphological characteristics of the capsules, and Fourier-transform infrared (FTIR) spectroscopy was used to evaluate their chemical properties. We found that SDMCs undergo bulk degradation in a species-dependent manner, with camellia SDMCs undergoing the most extensive degradation, and dandelion and lycopodium SDMCs being the most robust.

Carboxymethylpachymaran entrapped plant-based hollow microcapsules for delivery and stabilization of ß-galactosidase.

ß-Galactosidase (ß-Gal) as a dietary supplement can alleviate symptoms of lactose intolerance. However, ß-Gal is deactivated due to the highly acidic conditions and proteases in the digestive tract. In this work, ß-Gal was encapsulated into L. clavatum sporopollenin exine capsules (SECs) to fabricate an oral-controlled release system and increase the stability of ß-Gal in the digestive tract. The SEC extraction process was optimized. A 3-hour vacuum loading was determined as the optimal loading time. Five different initial ratios of SECs : ß-Gal were optimized with the maximum enzyme retention rate reaching 79.40 ± 1.96%. Furthermore, ß-Gal-loaded SECs entrapped in carboxymethylpachymaran (CMP) could control the release of ß-Gal under simulated gastrointestinal conditions (SGC). The optimal enzyme retention rate reached 65.33 ± 1.46% within 24 h under SGC. Collectively, these results indicated that the entrapped SECs could be used as an effective oral delivery vehicle of ß-Gal to improve its performance as a dietary supplement in the digestion of lactose.

Investigation of the Fate of Proteins and Hydrophilicity/Hydrophobicity of Lycopodium clavatum Spores after Organic Solvent-Base-Acid Treatment.

Microcapsules extracted from lycopodium ( Lycopodium clavatum) spores have been increasingly used as an oral therapeutic carrier. A series of sequential treatments involving acetone, KOH, and H3PO4 are used to extract a protein-free hollow microcapsule. This study focuses on two critical aspects of lycopodium spores: the fate of native proteins and the wettability of the spores after a chemical treatment. Protein-free spores are desired to prevent an allergic reaction, whereas the wettability is critical for the formulation development. Although the chemically treated lycopodium spores are generally regarded as protein free, the studies that have reported this have not gone into significant depths to understand the nature of residual nitrogen observed even in spores thought to be protein free. Wettability of spores has not received any significant attention. Accordingly, in this study, we performed a comprehensive analysis of natural spores and spores after each chemical treatment step. We show that natural lycopodium spores are hydrophobic and contain low-molecular-weight proteins (∼10 kD). Acetone treatment partially solubilizes unsaturated phospholipids from the spores. Nevertheless, the acetone-treated spores retain native proteins and are still hydrophobic. KOH treatment, however, removes a significant amount of proteins and partially hydrolyzes esters to carboxylic acid salts and results in a hydrophilic spore with a good wettability. Finally, we show that the H3PO4 treatment removes residual proteins, hydrolyzes remaining esters to carboxylic acids, and dissolves carbohydrates. H3PO4 treatment temperature controls carbohydrate dissolution, which in turn affects the hydroxyl functional groups and hydrophilicity (wettability) of the treated spores. Spores treated at 60 °C as opposed to 160 °C are amphiphilic in nature due to the abundance of hydroxyl functional groups on the surface. In conclusion, this study confirms the removal of native proteins from treated spores and sheds light on the chemical changes that the spores undergo after chemical treatment and correlates these changes to their wettability.

Ecophysiology and lipid dynamics of a eukaryotic mangrove decomposer.

Aurantiochytrium limacinum is an osmo-heterotrophic Stramenopile and a pioneering mangrove decomposer which is taxonomically assigned to the family of Thraustochytriaceae (class: Labyrinthulomycetes). The life cycle of A. limacinum involves different cell types including mono- and multi-nucleated cells as well as flagellated zoospores which colonize new fallen leaves. The ecological relevance of thraustochytrids is underestimated and eclipsed by their biotechnological importance, due to their ability to accumulate large amount of lipids, mainly triacylglycerols (TAGs). In this study, we aimed to understand the ecophysiological parameters that trigger zoospore production and the interplay between the life cycle of A. limacinum and its lipid metabolism. When grown in a rich medium, cells accumulated large amounts of TAGs at the end of their growth period, but no zoospores were produced. In poor media such as artificial sea water, zoospores were produced in massive quantities. In the absence of organic carbon, the zoospores remained swimming for at least 6 days, consuming their TAGs in the process. Addition of glucose rapidly triggered the maturation of the zoospores. On the basis of these data, we propose a life cycle for A. limacinum integrating the potential perturbations/changes in the environment surrounding a mangrove leaf that could lead to the production of zoospores and colonization of new areas.

Topographic cues guide the attachment of diatom cells and algal zoospores.

Surface topography plays a key role in the colonization of substrata by the colonizing stages of marine fouling organisms. For the innovation of marine antifouling coatings, it is essential to understand how topographic cues affect the settlement of these organisms. In this study, tapered, spiked microstructures and discrete honeycombs of varying feature dimensions were designed and fabricated in order to examine the influence of topography on the attachment of zoospores of the green macroalga Ulva linza and cells of the diatom (microalga) Navicula incerta. Contrasting results were obtained with these two species of algae. Indeed, the preferred location of cells of N. incerta was dominated by attachment point theory, which suggested a positive correlation between the density of cells adhering and the amount of available attachment points, while the settlement of spores of U. linza was mainly regulated by both Wenzel roughness and local binding geometry.

Production of spore laccase from Bacillus pumilus W3 and its application in dye decolorization after immobilization.

Given that spore laccase from the Bacillus genus is heat- and alkali-resistant, it is more suitable for industrial applications than fungal laccase. To determine the optimal culture conditions for spore laccase production, the effects of Cu2+ concentration, oxygen content, and culture time on spore laccase production from Bacillus pumilus W3 were investigated. The optimal production parameters were 0.2 mM of Cu2+, 200 rpm shaking speed, 100 mL liquid loading, and 5 days of cultivation. Spore laccase was efficiently immobilized on amino-functionalized celite. When used in dye decolorization, the immobilized spore laccase removed 84.15% of methyl green and 69.70% of acid red 1 after 48 h of treatment. Moreover, the immobilized spore laccase retained 87.04% of its initial decolorization activity after six cycles in the decolorization of acid red 1. These insights into the culture conditions and immobilization of spore laccases should be useful in the development of spore laccase as a biocatalyst in the treatment of textile wastewater.

Synergistic Activity Between S-Layer Protein and Spore-Crystal Preparations from Lysinibacillus sphaericus Against Culex quinquefasciatus Larvae.

Lysinibacillus sphaericus is used for the biological control of mosquitoes. The main toxicity mechanism of pathogenic strains is a binary toxin produced during sporulation. S-layer is a proteinaceous structure on the surface of bacteria; its functions have been involved in the interaction between bacterial cells and the environment, for example, as protective coats, surface recognition, and biological control. In L. sphaericus, S-layer protein (SlpC) is expressed in vegetative cells, and is also found in spore-crystal preparations; it has larvicidal activity in Culex spp. In this study, partial and completed sporulated culture toxicities were compared; also, S-layer protein and spore-crystal proteins were tested against Culex quinquefasciatus larvae for possible interactions. Larvicidal activity obtained with a combination of SlpC and spore-crystal proteins from strain III(3)7 showed no significant interaction, whereas, combinations of both preparations from strain 2362 showed synergistic effect. The highest synergistic activity observed was between spore protein complex from strain 2362 and SlpC from III(3)7. S-layer protein could be considered a good alternative in formulation improvement, for biological control of mosquitoes.

An autonomous lab on a chip for space flight calibration of gravity-induced transcellular calcium polarization in single-cell fern spores.

This report describes the development of lab-on-a-chip device designed to measure changes in cellular ion gradients that are induced by changes in gravitational (g) forces. The bioCD presented here detects differential calcium ion concentrations outside of individual cells. The device includes sufficient replicates for statistical analysis of the gradients around multiple single cells and around control wells that are empty or include dead cells. In the data presented, the degree of the cellular response correlates with the magnitude of the g-force applied via rotation of the bioCD. The experiments recorded the longest continuous observation of a cellular response to hypergravity made to date, and they demonstrate the potential utility of this device for assaying the threshold of cells' g-force responses in spaceflight conditions.

Detection of chlorophylls in spores of seven ferns.

Fern spores were traditionally classified into chlorophyllous (green) and nonchlorophyllous (nongreen) types based on the color visible to the naked eye. Recently, a third type, "cryptochlorophyllous spores", is recognized, and these spores are nongreen under white light but contain chlorophylls. Epifluorescence microscopy was previously used to detect chlorophylls in cryptochlorophyllous spores. In addition to epifluorescence microscopy, current study performed some other approaches, including spore-squash epifluorescence, absorption spectra, laser-induced fluorescence emission spectra, thin layer chromatography (TLC), and ultra-high performance liquid chromatography with ultraviolet and mass spectrometric detection (UHPLC-UV-MS) in order to detect chlorophylls of spores of seven ferns (Sphaeropteris lepifera, Ceratopteris thalictroides, Leptochilus wrightii, Leptochilus pothifolius, Lepidomicrosorum buergerianum, Osmunda banksiifolia, and Platycerium grande). Destructive methods, such as TLC and UHPLC-UV-MS, successfully detected chlorophylls inside the spores when their signals of red fluorescence under epifluorescence microscope were masked by spore wall. Although UHPLC-UV-MS analysis was the most sensitive and reliable for determining the chlorophylls of spores, spore-squash epifluorescence is not only reliable but also cost- and time-effective one among our study methods. In addition, we first confirmed that Lepidomicrosorium buergerianum, Leptochilus pothifolius, Leptochilus wrightii, and Platycerium grande, produce cryptochlorophyllous spores.

Macroporous natural capsules extracted from Phoenix dactylifera L. spore and their application in oral drugs delivery.

Macroporous natural sporopollenin exine capsules (SEC) were extracted from date palm (Phoenix dactylifera L.) and coated by natural polymer composite (carboxymethyl cellulose with epichlorohydrin). The polymer coated exine capsules (PCEC) were used in in-vitro investigations for controlled delivery of paracetamol. SEC, PCEC, and drugs loaded capsules (PCEC-PAR) were characterized by scanning electron microscope (SEM), surface area (BET), Fourier-transform infrared (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The length of SEC was found to be 20-20.5 µm, and the pore sized was 50-135 nm, as measured using SEM. The studies revealed that maximum loading of the drug was at pH 6.0 (97.2%, with 50 mg mL(-1)). The results indicate that by increasing the pH from 1.4 to 7.4, the cumulative release rates of paracetamol in physiological buffer solution (PBS) is more than two times as in simulated gastric fluid (SGF). In addition, the in-vitro toxicity of PCEC against Caco-2 cells was tested by the 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay, and the results revealed that PCEC are biocompatible materials. The overall results encourage further studies on the clinical use of PCEC as drug carriers.

Hematotoxicity and genotoxicity evaluations in Swiss mice intraperitoneally exposed to Bacillus thuringiensis (var kurstaki) spore crystals genetically modified to express individually Cry1Aa, Cry1Ab, Cry1Ac, or Cry2Aa.

Bacillus thuringiensis (Bt) has been widely used in foliar sprays as part of integrated pest management strategies against insect pests of agricultural crops. Since the advent of genetically modified plants expressing Bt δ-endotoxins, the bioavailability of Cry proteins has increased, and therefore for biosafety reasons their adverse effects should be studied, mainly for nontarget organisms. We evaluated, in Swiss mice, the hematotoxicity and genotoxicity of the genetically modified strains of Bt spore crystals Cry1Aa, 1Ab, 1Ac, or 2Aa at 27 mg/kg, and Cry1Aa, 1Ab and 2Aa also at 136 and 270 mg/kg, administered with a single intraperitoneal injection 24 h before euthanasia. Controls received filtered water or cyclophosphamide. Blood samples collected by cardiac puncture were used to perform hemogram, and bone marrow was extracted for the micronucleus test. Bt spore crystals presented toxicity for lymphocytes when in higher doses, which varied according to the type of spore crystal studied, besides promoting cytotoxic and genotoxic effects for the erythroid lineage of bone marrow, mainly at highest doses. Although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results indicated that these Bt spore crystals were not harmless to mice. This suggests that a more specific approach should be taken to increase knowledge about their toxicological properties and to establish the toxicological risks to nontarget organisms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 970-978, 2016.

[Morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius: a contribution to the systematics of Neotropical Lycopodiaceae]. / Morfología y anatomía de ejes caulinares, licofilos y esporangios de Phlegmariurus phylicifolius: un aporte a la sistemática de las Lycopodiaceae neotropicales.

Phlegmariurus is the only genus of Lycopodiaceae with the species grouped in 22 informal groups. Species level relationships within Phlegmariurus are poorly understood and their circumscriptions require a thorough molecular and morphological review. A detailed study of morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius was carried out in order to contribute to the elucidation of species circumscription in the informal group Phlegmariurus phlegmaria. Small pieces of caulinar axes bearing trophophylls, sporophylls and sporangia were fixed, dehydrated, Histowax (paraffin) embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made and stained with the same technique. P. phylicifolius includes slender, pendulous plants up to 40cm long. Shoots heterophyllous, in the basal divisions ca. 10-20(-25) mm in diameter including the trophophylls, then abruptly constricted to (1-) 1.5-2(-2.5) mm in diameter including the imbricate, reduced sporophylls. Trophophylls are borne in alternating whorls of three, or decussate, subdecussate, or alternate, widely spaced in alternate leaved caulinar axes portions, perpendicular to the caulinar axes to falcately ascending, lanceolate to linear-lanceolate, with flat to slightly revolute margins. Each lycophyll is supplied by a single central vascular bundle, connected to a protoxylem pole in the stele. At the site of leaf-trace departure, no leaf (lycophyll) gap is present. Caulinar axes excluding leaves 0.7-1.2 mm thick at the base, upward tapering to ca. 0.5 mm. Caulinar axes present unistratified epidermis and endodermis, the cortex is characterized by the presence of a trabecular structure of lisigenous origin formed in the parenchimatous tissue next to the endodermis. The vascular tissue occupies the central part of the caulinar axes, forming a plectostele ofsubradiate organization, with five poles ofprotoxylem. The epidermal cells present sinuous anticlinal walls; invaginations in the inner side of external periclinal wall of the epidermal cells could be probably adaptive morphological feature of a water deficient environment. Leaves of constricted terminal divi- sions are decussate, or subdecussate, continuously or discontinuously sporangiate, appressed, abaxially rounded to carinate, widely lanceolate to widely ovate or subcordate, acute to mucronate or cuspidate, shorter than the sporangia. Each sporangium originates from a group of epidermal cells, axilar to the sporophylls. The cell walls of epidermal cell of the sporangia are Huperzioideae type. The morphological studies of trophophylls contribute to confirm the differences between P. phylicifolius and P. subulatus.