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.

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.

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.

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

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 (l-) 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 0.7-1.2mm thick at the base, upward tapering to ca. 0.5mm. 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 of subradiate organization, with five poles of protoxylem. 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 divisions 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. Rev. Biol. Trop. 62 (3): 1217-1227. Epub 2014 September 01.

Phlegmariurus es el único género de Lycopodiaceae con las especies reunidas en 22 grupos informales. Las relaciones a nivel de especie dentro de Phlegmariurus están pobremente estudiadas y la circunscripción de las mismas requiere profundos exámenes moleculares y morfológicos. Se ha llevado a cabo un estudio detallado de la morfología y la anatomía de ejes caulinares, licofilos y esporangios de P. phylicifolius, con el fin de contribuir al esclarecimiento en la delimitación de las especies en el grupo Phlegmariurus phlegmaria. Segmentos de ejes caulinares con trofofilos, esporofilos y esporangios fueron fijados, deshidratados, incluidos en Histowax (parafina), cortados con un micrótomo rotatorio y coloreados usando la técnica tradicional Safranina O-Verde Rápido; además se hicieron cortes a mano alzada y se colorearon con la misma técnica. P. phylicifolius incluye plantas colgantes y péndulas de hasta 40cm de longitud. Los ejes son heterofilos, de aproximadamente 10-20(-25)mm de diámetro en las divisiones basales incluyendo los trofofilos, luego abruptamente reducidos a (l-) 1.5-2(-2.5)mm de diámetro incluyendo los esporofilos reducidos e imbricados. Los trofofilos están dispuestos en anillos alternantes de a tres, o decusados, subdecusados o alternos, dispuestos en forma espaciada en los ejes caulinares, perpendiculares al tallo hasta falcadamente ascendentes, lanceolados a lineal-lanceolados, con márgenes lisos o levemente revolutos. Cada licofilo está provisto de un haz vascular simple y central, conectado a un polo de protoxilema de la estela y sin laguna foliar. Los tallos poseen un ancho de 0.7-1.2mm en la base, excluyendo los licofilos, estrechándose hasta cerca de 0.5mm hacia el ápice. Los ejes caulinares presentan una epidermis uniestratificada y endodermis, la corteza se caracteriza por la presencia de una estructura trabecular de origen lisígeno formada en el tejido parenquimático próximo a la endodermis. El tejido vascular ocupa la parte central del eje caulinar, formando una plectostela de organización subradiada, con cinco polos de protoxilema. Las células epidérmicas presentan paredes anticlinales sinuosas; las invaginaciones en la cara interna de la pared periclinal externa podrían ser probablemente un característica morfológica adaptativa a un ambiente con períodos de sequía. Las hojas de las porciones apicales son decusadas o subdecusadas, con esporangio de disposición continua o discontinua, adpresas, abaxialmente redondeadas a carinadas, ampliamente lanceoladas a ovadas o subcordadas, ápice agudo a mucronado o cuspidado, más corto que el esporangio. Cada esporangio se origina de un grupo de células epidérmicas, en la axila de los esporofilos con el eje caulinar. Las paredes celulares de las células epidérmicas del esporangio son de tipo Huperzioideae. El estudio de la morfología de los trofofilos contribuye a confirmar las diferencias entre P. phylicifolius y P. subulatus.

The inhibitory effect of an ethanol extract of the spores of Lygodium japonicum on ethylene glycol-induced kidney calculi in rats.

We investigated the effect of an ethanol extract of Lygodii spora (LS) as a preventive and therapeutic agent for experimentally induced calcium oxalate nephrolithiasis with ethylene glycol (EG) in rats. Male Wistar rats were randomly divided into preventive (n = 18, for 28 days) and therapeutic (n = 24, for 42 days) groups. The preventive group was further subdivided into three groups of six rats each: preventive control, preventive lithiatic control (EG) and preventive lithiatic LS (EG + 400 mg/kg LS). Similarly, the therapeutic group was subdivided into four groups of six rats each: therapeutic control, therapeutic lithiatic control, therapeutic lithiatic untreated, and therapeutic lithiatic LS. Lithiasis was induced by adding 0.75% EG to the drinking water of all groups except the preventive and therapeutic control groups. Preventive and therapeutic subjects also received the LS ethanol extract in drinking water at a dose of 400 mg/kg, since day 0 or day 28, respectively. At the end of the each experimental period, various biochemical parameters were measured in urine and kidney homogenates. The kidneys were subjected to histopathological analysis. The results revealed that treatment with the LS preventive protocol significantly decreased the levels of urinary calcium, oxalate and uric acid, and increased the levels of urinary citrate as compared to those in the EG control. No significant changes in the urinary parameters except oxalate and citrate levels were observed in the rats in the therapeutic protocol. In both preventive and therapeutic protocols, the extract significantly decreased kidney peroxides, renal calcium, oxalate content, and the number of kidney oxalate deposits as compared to those in the EG group. We conclude that LS is useful as a preventive and therapeutic agent against the formation of oxalate kidney stones.

Ontogenia de los esporangios, formación y citoquímica de esporas en licopodios (Lycopodiaceae) colombianos / Ontogeny of the sporangium, spore formation and cytochemistry in Colombian Lycopodials (Lycopodiaceae)

Studies on reproductive aspects of Lycopodiaceae are not very abundant in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. Here we present a detailed study of the ontogeny of sporangia and sporogenesis, and the chemical determination of several compounds generated during spore formation. The analyses were performed in 14 taxa of six genera of the family, Diphasiastrum, Diphasium, Huperzia (a genus which is treated here including Phlegmariurus), Lycopodiella, Lycopodium and Palhinhaea. Specimens were collected in three departments from the Colombian Andes between 1 454-3 677m altitude. Ontogeny was studied in small, 1cm long pieces of strobili and axis, which were fixed in glutaraldehyde or FAA, dehydrated in alcohol, embedded in LR White, sectioned in 0.2-0.5μm and stained with toluidine blue (TBO), a metachromatic dye that allows to detect both sporopollenin and lignin or its precursors, during these processes. For other studies, paraplast plus-embedded sections (3-5μm) were stained with safranin-fast green and alcian blue-hematoxylin. Chemical tests were also conducted in sections of fresh sporangia at different stages of maturity using alcian blue (mucopolysaccharides), Lugol solution (starch), Sudan III (lipids), phloroglucinol (lignin) and orcein (chromosomes). Sections were observed with photonic microscope equipped with differential interference contrast (DIC) and fluorescence microscopy (for spore and sporangium walls unstained). Strobili and sporangia were dehydrated with 2.2 dimethoxypropane, critical point dried and coated with gold for scanning electron microscopy (SEM). Our results indicated that the ontogeny of sporangia and sporogenesis were very similar to the previously observed in Huperzia brevifolia. Cutinisation occurs in early stages of development of sporangium cell walls, but in their final stages walls become lignified. As for the sporoderm development, the exospore is the first layer formed, composed by sporopollenin. The endospore deposits as a thin inner layer composed of cellulose, pectin and carboxylated polysaccharides. The perispore, if present, deposits at last. Mucopolysaccharides were found on the sporocyte coat and its abundance in sporangial cavity persists up to the immature tetrads stage, and then disappears. The lipids were abundant in the sporocytes, tetrads and spores, representing the main source of energy of the latter. In contrast, starch is not detected in the spores, but is abundant in premeiotic sporocytes and immature tetrads, developmental stages of high cellular metabolic activity. Intrinsic fluorescence corroborates the presence of lignin and cutin in the sporangium wall, while the sporopollenin is restricted to the exospore. The transfusion cells and the perispore are not always present. However, the processes of ontogeny and sporogenesis are extremely similar throughout the taxa studied, suggesting that they represent conservative family traits, nonspecific or generic.

Los estudios sobre aspectos reproductivos no son muy abundantes en la literatura científica sobre los taxones de Lycopodiaceae y constituyen información esencial para apoyar la taxonomía y relaciones sistemáticas en el grupo. Por lo tanto, se presenta aquí un análisis detallado de la ontogenia de los esporangios y esporogénesis, así como determinaciones químicas de varios compuestos generados durante la formación de las esporas. Los análisis se llevaron a cabo en 14 taxones de seis géneros de la familia: Diphasiastrum, Diphasium, Huperzia (un género que se trata aquí, incluyendo Phlegmariurus), Lycopodiella, Lycopodium y Palhinhaea. Las muestras fueron recolectadas en tres departamentos de los Andes de Colombia entre 1 454-3 677m de altitud. La ontogenia se estudió en trozos de estróbilos y ejes, de 1cm de largo, que se fijaron en glutaraldehido o FAA, se deshidrataron en alcohol, se incluyeron en LR White, se seccionaron en cortes de 0.2-0.5μm y se colorearon con azul de toluidina (TBO), un colorante metacromático que permite detectar tanto esporopolenina como lignina o sus precursores. Para estudios adicionales, secciones de 3-5μm de material incluido en paraplast plus se colorearon con safranina-verde rápido y azul alciánhematoxilina. Las pruebas químicas se llevaron a cabo en secciones de esporangios sin fijar en diferentes etapas de madurez utilizando azul alcián (mucopolisacáridos), solución de Lugol (almidón), Sudán III (lípidos), fluoroglucinol (lignina) y orceína (cromosomas). Las observaciones se efectuaron con microscopio fotónico equipado con contraste diferencial de interferencia (DIC) y microscopía de fluorescencia (para esporas y pared de los esporangios sin colorear). Para observaciones con microscopía electrónica de barrido (MEB), los estróbilos y esporangios se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Los resultados indican que la ontogenia de los esporangios y esporogénesis es muy similar a la observada previamente en Huperzia brevifolia. En las primeras etapas de desarrollo, las paredes celulares de la epidermis del esporangio se cutinizan y en las finales se lignifican. En el desarrollo del esporodermo, la primera capa que se forma es el exosporio, compuesto por esporopolenina. El endosporio es una capa interna delgada compuesta de celulosa, pectina y polisacáridos carboxilados. El perisporio, si está presente, es la última capa que se deposita. Los mucopolisacáridos se encontraron en la cubierta del esporocito, son abundantes en la cavidad esporangial hasta la etapa de tétradas inmaduras y luego desaparecen. Los lípidos son abundantes en esporocitos, tétradas y esporas, y representan la principal fuente de energía de estas. En contraste, el almidón no se detecta en las esporas pero es abundante en esporocitos premeióticos y tétradas inmaduras, ambos con gran actividad metabólica. La fluorescencia intrínseca corrobora la presencia de lignina y cutina en la pared del esporangio, mientras que la esporopolenina se limita al exosporio. Las células de transfusión y el perisporio no siempre están presentes. Sin embargo, los procesos de la ontogenia y esporogénesis son extremadamente similares en todos los taxones estudiados, lo que sugiere que representan rasgos típicos de familia, no específicos ni genéricos.

Ptaquiloside in bracken spores from Britain.

Secondary metabolites from bracken fern (Pteridium aquilinum (L.) Kuhn) are suspected of causing cancer in humans. The main carcinogen is the highly water-soluble norsesquiterpene glucoside ptaquiloside, which may be ingested by humans through food, e.g. via contaminated water, meat or milk. It has been postulated that carcinogens could also be ingested through breathing air containing bracken spores. Ptaquiloside has not previously been identified in bracken spores. The aim of the study was to determine whether ptaquiloside is present in bracken spores, and if so, to estimate its content in a collection of spores from Britain. Ptaquiloside was present in all samples, with a maximum of 29 µg g(-1), which is very low compared to other parts of the fern. Considering the low abundance of spores in breathing air under normal conditions, this exposure route is likely to be secondary to milk or drinking water.

A new species of Myxozoa, Henneguya rondoni n. sp. (Myxozoa), from the peripheral nervous system of the Amazonian fish, Gymnorhamphichthys rondoni (Teleostei).

Henneguya rondoni n. sp. found in the peripheral lateral nerves located below the two lateral lines of the fish Gymnorhamphichthys rondoni (Teleostei, Rhamphichthyidae) from the Amazon river is described using light and electron microscopy. Spherical to ellipsoid cysts measuring up to 110 microm in length contained only immature and mature spores located in close contact with the myelin sheaths of the nervous fibres. Ellipsoidal spores measured 17.7 (16.9-18.1)-microm long, 3.6 (3.0-3.9)-microm wide, and 2.5 (2.2-2.8)-microm (n=25) thick. The spore body measuring 7.0 (6.8-7.3)-microm long was formed by two equal symmetric valves, each with an equal tapering tail 10.7 (10.3-11.0) microm in length. The tails were composed of an internal dense material surrounded by an external homogeneous sheath of hyaline substance. The valves surrounded two equal pyriform polar capsules measuring 2.5 (2.2-2.8)-microm long and 0.85 (0.79-0.88)-microm (n=25) wide and a binucleated sporoplasm cell containing globular sporoplasmosomes 0.38 (0.33-0.42) microm (n=25) in diam. with an internal eccentric dense structure with half-crescent section. Each polar capsule contains an anisofilar polar filament with 6-7 turns obliquely to the long axis. The matrix of the polar capsule was dense and the wall filled with a hyaline substance. The spores differed from those of previously described species. Based on the ultrastructural morphology of the spore and specificity to the host species, we propose a new species name H. rondoni n. sp.

Rapid determination of spore chemistry using thermochemolysis gas chromatography-mass spectrometry and micro-Fourier transform infrared spectroscopy.

Spore chemistry is at the centre of investigations aimed at producing a proxy record of harmful ultraviolet radiation (UV-B) through time. A biochemical proxy is essential owing to an absence of long-term (century or more) instrumental records. Spore cell material contains UV-B absorbing compounds that appear to be synthesised in variable amounts dependent on the ambient UV-B flux. To facilitate these investigations we have developed a rapid method for detecting variations in spore chemistry using combined thermochemolysis gas chromatography-mass spectrometry and micro-Fourier transform infrared spectroscopy. Our method was tested using spores obtained from five populations of the tropical lycopsid Lycopodium cernuum growing across an altitudinal gradient (650-1981 m a.s.l.) in S.E. Asia with the assumption that they experienced a range of UV-B radiation doses. Thermochemolysis and subsequent pyrolysis liberated UV-B pigments (ferulic and para-coumaric acid) from the spores. All of the aromatic compounds liberated from spores by thermochemolysis and pyrolysis were active in UV-B protection. The various functional groups associated with UV-B protecting pigments were rapidly detected by micro-FTIR and included the aromatic C[double bond, length as m-dash]C absorption band which was exclusive to the pigments. We show increases in micro-FTIR aromatic absorption (1510 cm(-1)) with altitude that may reflect a chemical response to higher UV-B flux. Our results indicate that rapid chemical analyses of historical spore samples could provide a record ideally suited to investigations of a proxy for stratospheric O3 layer variability and UV-B flux over historical (century to millennia) timescales.

[Ganoderma spores may regulate the levels of mitochondria-related molecular substances in hippocampus of young rats birthed by rats with gestational hypertension].

OBJECTIVE: To investigate the effects of Ganoderma spores on mitochondria-related molecular substances in hippocampus of young rats birthed by rats with gestational hypertension. METHODS: Nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methylester (L-NAME) was intraperitoneally injected into pregnant rats to induce gestational hypertension, and Ganoderma spores were administered orally. The effects of Ganoderma spores on levels of mitochondria-related molecular substances in hippocampus of young rats birthed by the rats with gestational hypertension were evaluated with immunoradiometric assay of cAMP, RT-PCR analysis of related genes, and detection of enzyme activity. RESULTS: In hippocampus of the new-born rats birthed by rats with gestational hypertension, the cAMP level, mitochondrial DNA (mtDNA) level and adenosine triphosphatase (ATPase) activity were decreased, and the expression level of peroxisome proliferator activated receptor gamma coactivator 1 alpha (pgc1 alpha) was unchanged compared to the normal control group. The cAMP level, mtDNA level, ATPase activity and pgc1 alpha expression level in hippocampus of 30-day post-natal rats were lower than those of the rats in normal control group. After oral administration of Ganoderma spores, the cAMP and mtDNA levels in hippocampus of the new-born rats and 30-day post-natal rats recovered almost to the levels of normal control rats, and the ATPase activity and pgc1 alpha expression level were also increased significantly. CONCLUSION: Ganoderma spores may regulate the levels of mitochondria-related molecular substances in hippocampus of young rats birthed by rats with gestational hypertension.

[Effect of the oil from ganoderma lucidum spores on pathological changes in the substantia nigra and behaviors of MPTP-treated mice].

OBJECTIVE: To investigate the oil from the spores of ganoderma lucidum, a rare Chinese herb, on the behaviors and pathological changes in the substantia nigra pars compacta (SNpc) in mouse models of Parkinson's disease (PD) induced by MPTP. METHODS: C57BL mice were divided into 3 groups, and the ganoderma spores oil + MPTP group were treated with ganoderma spores oil for 8 days, together with subcutaneous injection of MPTP (30 mg/kg) starting on the third day for 6 days; MPTP group were pretreated with normal saline before subcutaneous MPTP injection, and the normal control group received pretreatment with normal saline before subcutaneous normal saline injection. The behavioral changes of the mice in different groups were observed by pole test, dopamine and its metabolic products in the striatum determined by HPLC, tyrosine hydroxylase (TH) positive cells detected by immunofluorescence method, and expression of TH protein by Western blotting. RESULTS: The mice in the ganoderma spores oil + MPTP group presented significantly less involuntary movement of the limbs in the pole test than the mice in MPTP group. The levels of dopamine and DOPAC in the striatum of ganoderma spores oil-treated mice were increased as compared with those in MPTP group. The number of surviving TH-positive neurons in SNpc of mice in ganoderma spores oil + MPTP group was significantly greater than that in MPTP group, with also significantly increased TH protein expression. CONCLUSION: Ganoderma spores oil has neuroprotective effect for preventing doparminergic neuron from impairment by MPTP.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in clinical chemistry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-Tof-MS) has recently become a popular and versatile method to analyze macromolecules from biological origin. In this paper, we will review the application of MALDI-Tof-MS in clinical chemistry and biology. MALDI-Tof-MS is used in clinical chemistry, e.g. disease markers can be identified with MALDI-MS analysis in combination with 1-D and 2-D gel electrophoresis separations thanks to either peptide mass fingerprinting (PMF) or peptide sequence tag (PST) followed by data base searching. In microbiology, MALDI-Tof-MS is employed to analyze specific peptides or proteins directly desorbed from intact viruses, bacteria and spores. The capability to register biomarker ions in a broad m/z range, which are unique and representative for individual microorganisms, forms the basis of taxonomic identification of bacteria by MALDI-Tof-MS. Moreover, this technique can be applied to study either the resistance of bacteria to antibiotics or the antimicrobial compounds secreted by other bacterial species. More recently, the method was also successfully applied to DNA sequencing (genotyping) as well as screening for mutations. High-throughput genotyping of single-nucleotide polymorphisms has the potential to become a routine method for both laboratory and clinical applications. Moreover, posttranscriptional modifications of RNA can be analyzed by MALDI using nucleotide-specific RNAses combined with further fragmentation by post source decay (PSD).

Legumin- and vicilin-like proteins from spores of the fern Matteuccia struthiopteris.

Legumin- and vicilin-like proteins have been isolated from spores of the fern Matteuccia struthiopteris. Their relationship with seed legumin and vicilin was demonstrated by cross-reactivities of antibodies directed against respective storage globulins from Vicia faba as evidenced by Western blotting. The Matteuccia legumin-like protein was characterised as a 300-340 kDa holoprotein preferentially consisting of a 32 kDa alpha-chain and a 24 kDa beta-chain. Patterns of limited proteolysis of the spore legumin-like protein and seed legumins were similar as well. In contrast to seed legumins, the Matteuccia legumin-like protein is devoid of disulfide bridges between alpha- and beta-chains. A 52 kDa polypeptide of the Matteuccia vicilin-like protein, first detected by SDS gel electrophoresis, is probably encoded by a vicilin-like gene specifically expressed in Matteuccia struthiopteris spores (Shutov et al. 1998). The vicilin-like holoprotein was found to form a complex of 600 kDa apparent molecular mass, presumably composed of four vicilin-like trimers.

Lipids of three microsporidian species and multivariate analysis of the host-parasite relationship.

Sporal lipids of 3 microsporidia, Encephalitozoon cuniculi from mammals and Glugea atherinae and Spraguea lophii from fishes, were investigated. High phospholipid levels were found (54.8-64.5% of total lipids), which is in agreement with the presence of highly developed internal membranes in microsporidian spores. Sphingomyelin was not detected in G. atherinae. Triglycerides (less than 10% of total lipids), cholesterol, and free fatty acids were identified in all species. Analysis of fatty acids from the phospholipid fraction revealed the predominance of docosahexaenoic acid (30-40% of total phospholipid fatty acids) in G. atherinae and S. lophii and oleic acid (25.8% of total phospholipid fatty acids) in E. cuniculi. The 3 microsporidia possessed a significant amount of branched-chain fatty acids (iso and anteiso forms) not found in the hosts, supporting the existence of some parasite-specific metabolic steps for these fatty acids. On the basis of phospholipid fatty acid profiles, host-parasite relationships were investigated through correspondence factorial analysis. It shows 3 distinct clusters with the first corresponding to fishes, the second to fish parasites, and the third to E. cuniculi and its host cell. These data suggest that the mammal microsporidia developing within parasitophorous vacuoles are more dependent on host cells than the fish microsporidia that induce cystlike structures.

Triterpenes from the spores of Ganoderma lucidum and their cytotoxicity against meth-A and LLC tumor cells.

Six new highly oxygenated lanostane-type triterpenes, called ganoderic acid gamma (1), ganoderic acid delta (2), ganoderic acid epsilon (3), ganoderic acid zeta (4), ganoderic acid eta (5) and ganoderic acid theta (6), were isolated from the spores of Ganoderma lucidum, together with known ganolucidic acid D (7) and ganoderic acid C2 (8). Their structures of the new triterpenes were determined as (23S)-7beta,15alpha,23-trihydroxy-3,11-dioxolanosta-8, 24(E)-diene-26-oic acid (1), (23S)-7alpha,15alpha23-trihydroxy-3,11-dioxolanosta-8, 24(E)-diene-26-oic acid (2), (23S)-3beta3,7beta, 23-trihydroxy-11,15-dioxolanosta-8,24(E)-diene-26-oic acid (3), (23S)-3beta,23-dihydroxy-7,11,15-trioxolanosta-8, 24(E)-diene-26-oic acid (4), (23S)-3beta,7beta,12beta,23-tetrahydroxy-11,15-dioxolanos ta-8,24(E)-diene-26-oic acid (5) and (23S)-3beta,12beta23-trihydroxy-7,11,15-trioxolanosta-8,24(E )-diene-26-oic acid (6), respectively, by chemical and spectroscopic means, which included the determination of a chiral center in the side chain by a modification of Mosher's method. The cytotoxicity of the compounds isolated from the Ganoderma spores was carried out in vitro against Meth-A and LLC tumor cell lines.

Fern spore extracts can damage DNA.

The carcinogenicity of the vegetative tissues of bracken fern (Pteridium) has long been established. More recently, the carcinogenic effects of the spores of bracken have also been recognized. Both vegetative tissues and spores of bracken can induce adducts in DNA in animal tissues, but the possible genotoxic or carcinogenic effects of spores from fern species other than bracken are unknown. The single-cell gel electrophoresis ('comet') assay was used to investigate whether fern spores can cause DNA damage in vitro. Extracts of spores from six fern species were administered to cultured human premyeloid leukaemia (K562) cells. Spore extracts of five fern species: Anemia phyllitidis, Dicksonia antarctica, Pteridium aquilinum, Pteris vittata and Sadleria pallida, induced significantly more DNA strand breaks than those in the control groups. Only in one species, Osmunda regalis, was the effect no different from that in the control groups. Using extracts from A. phyllitidis and P. vittata, the extent of DNA damage was increased by increasing the original dose 10 times, whereas an experiment in which exposure times were varied suggested that the highest levels of strand breaks appear after 2 h exposure. Simultaneous incubation with human S9 liver enzyme mix ablated the damaging effect of the extracts. Our data show that fern spore extracts can cause DNA damage in human cells in vitro. Considering the strong correlation between DNA damage and carcinogenic events, the observations made in this report may well have some implications for human health.

[Extraction and separation of antitumor components from Ganoderma lucidum (Leyss. ex Fr.) Karst].

OBJECTIVE: To examine the variation of alcohol extraction rates of Ganoderma lucidum spores with different rates of wall-wrack, and analyze the antitumor components of alcohol extract by chromatography. METHOD: The G. lucidum spores were soaked and extracted with absolute alcohol. The alcohol extract was chromatographed on a silica gel column and HPLC in proper order, and the antitumor activity of every eluted fraction was represented by its cytotoxicity towards Hela cells. RESULT: Extraction rates 5%, 25% and 33% corresponded to wall-wrack rates 0%, 60%-80% and 99% respectively. The alcohol extract from spores with the highest wall-wrack rate was chromatographed on a silica gel column, eluting successfully with CHCl3, EtOAc and CH3OH in order. The CHCl3 fraction had not any antitumor activity, while this activity of CH3OH fraction was 34 times greater than that of EtOAc fraction. HPLC analysis found out that two mixtures(II1 and II3) possess significant antitumor activity in vitro. CONCLUSION: The weight of alcohol extract from spores with wall-wrack was far greater than that of spores without. The antitumor components of G. lucidum spores could be analyzed with methanol-water on a reverse HPLC.

Soybean isoflavones trigger a calcium influx in Phytophthora sojae.

Both the motile zoospores and the hyphal germ tubes of Phytophthora sojae respond chemotropically to the soybean isoflavones daidzein and genistein. The role of Ca(2+) in the cellular response to these host signals was investigated by using X-ray microanalysis of cells to monitor net changes in cellular levels of Ca(2+) and by quantifying the effects of exogenous Ca(2+) and daidzein on the developmental fate of encysted zoospores. Confirmation that isoflavones trigger a net influx of Ca(2+) into the cell was demonstrated by X-ray microanalysis of individual encysted zoospores. Zoospores exposed to 10 mM Ca(2+) and 1 microM daidzein at the time of encystment formed cysts that contained more Ca(2+) than zoospores exposed to Ca(2+) alone. The magnitude of internal Ca(2+) stores appears to be a determining factor affecting the developmental fate of P. sojae cysts.

Transport of axl2p depends on erv14p, an ER-vesicle protein related to the Drosophila cornichon gene product.

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface. Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck. In erv14Delta strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates. We propose that Erv14p is required for the export of specific secretory cargo from the ER. The polarity defect of erv14Delta yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis. These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila.

Production of monoclonal antibodies against Nosema bombycis and their utility for detection of pebrine infection in Bombyx mori L.

Latex agglutination assay based on monoclonal antibodies (MCAs) described in this communication may be useful for detection of Pebrine infection in silkworm. Four murine MCAs were produced against Nosema bombycis spore. In ELISA all 4 MCAs (IgM isotype) reacted with alkali treated Nosema spores and to variable extent with acetone precipitated surface protein. However, MA-310 and MA-542 showed a low degree of cross reactivity with BmNPV. In contrast, MA-503 and MA-515 were devoid of reactivity with BmNPV, B. thuringiensis, S. marcescens, Azotobactor, Rhizobium and normal hemolymph protein in ELISA. Latex beads sensitized with a combination of MA-503 and MA-515 (50 micrograms each per ml of 0.4% latex beads) could detect 1 x 10(5) Nosema spores per test. Sensitization of the latex beads with the cocktail of these two MCAs through protein-A bridge further led to a 10-fold increase in the sensitivity (1 x 10(4) spores/test) of the assay. No agglutination was observed in presence of BmNPV, Rhizobium, Azotobactor, E. coli, B. thuringiensis, S. marcescens and normal hemolymph protein indicating the specificity of the test. The results obtained by latex agglutination assay on hemolymph samples of infected as well as normal larvae collected from field, II instar larvae infected in the laboratory and from infected mother moth revealed 100% correlation with results by microscopic examination.