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The Activation of Cyclophosphamide in the Hydra Developmental Toxicology Assay. Newman, L. M., JOHNSON, E. M., GIACOBBE, R. L., AND FU, L-J. (1990). . 15, 488–499. The proteratogen cyclophosphamide (CP) was tested in the Hydra Assay in the presence and absence of an metabolic activation package (MAP) consisting of rat hepatic microsomes (0.06 nmol P450/ml), 500 μM NADPH, and 25 μm MgCl. This metabolic system was developed through a series of interrelated biochemical and biological assays to provide maximum cytochrome P450 mixed-function monooxygenase (MFO) metabolic capacity while controlling the inherent toxicity of the hepatic preparation and the attendant cofactors. Bioactivation of CP was confirmed under standard hydra assay conditions of pH 7.0 and 20°C and compared with activation at 37°C. Estimation of total metabolic capacity and verification of activation were made through the appearance of alkylating metabolites both in the absence and in the presence of hydra. Chemical exposure was maintained throughout the 92 ± 2 hr assay with periodic renewal of media (and additives) at 4, 20, 28, 42, and 66 hr of incubation. Inclusion of bioactivation increased the toxicity of CP by two orders of magnitude. The minimal affective concentration in the adult and developmental portions of the assay was decreased from 4000 to 20 μg CP/ml and from 1000 to 4 μg CP/ml, respectively. By limiting the inherent toxicity of the MFO package, it was possible to avoid pulse-type exposures and ensure that all ontogenic stages were exposed to active metabolites. The addition of metabolic activation capacity to an assay, while not essential, markedly enhances its utility and breadth of application in developmental toxicity safety evaluations. [ABSTRACT FROM PUBLISHER]

Few studies have reported on polonium-210, a decay breakdown product of radon-222 and lead-210, in human lungs and there has been no study in patients with suspected lung cancer. The main aim of this "Polonium in vivo" study was to evaluate polonium-210 radioactivity in bronchopulmonary systems of smoker, ex-smoker and never smoker patients with suspected lung cancer. Alpha-spectrometric analyses were performed on bronchial lavage (BL) fluids from two Italian hospitals in 2013-2016. Socio-demographic, smoking, occupational and spirometric characteristics, lung cancer confirmation and histologic type and radon-222 concentration in patients' homes were collected. Seventy BL samples from never ( n = 13), former ( n = 35) and current smokers ( n = 22) were analyzed; polonium-210 was detected in all samples from current and former smokers and in 54% of samples from never smokers ( p < 0.001; median values: 1.20, 1.43 and 0.40 mBq, respectively). Polonium-210 levels were significantly higher in COPD versus no COPD patients (median value: 3.60 vs. 0.97 mBq; p = 0.007); former and current smokers, without and with COPD, had significantly increased polonium-210 levels ( p = 0.012); 96% of confirmed versus 69% of non-confirmed lung cancer patients recorded detectable polonium-210 levels ( p = 0.018). A polonium-210 detectable activity was measured in BL samples from all current and former smokers. Polonium-210 in the lungs could be the result of lead-210 entrapment, which, with its half-life of 22 years, could provide a continuous emission of alpha radioactivity, even many years after quitting, thus proposing a possible explanation for the onset of lung cancer, particularly in former smokers.

The emergency caused by Covid-19 pandemic raised interest in studying lifestyles and comorbidities as important determinants of poor Covid-19 prognosis. Data on tobacco smoking, alcohol consumption and obesity are still limited, while no data are available on the role of e-cigarettes and heated tobacco products (HTP). To clarify the role of tobacco smoking and other lifestyle habits on COVID-19 severity and progression, we designed a longitudinal observational study titled COvid19 and SMOking in ITaly (COSMO-IT). About 30 Italian hospitals in North, Centre and South of Italy joined the study. Its main aims are: 1) to quantify the role of tobacco smoking and smoking cessation on the severity and progression of COVID-19 in hospitalized patients; 2) to compare smoking prevalence and severity of the disease in relation to smoking in hospitalized COVID-19 patients versus patients treated at home; 3) to quantify the association between other lifestyle factors, such as e-cigarette and HTP use, alcohol and obesity and the risk of unfavourable COVID-19 outcomes. Socio-demographic, lifestyle and medical history information will be gathered for around 3000 hospitalized and 700-1000 home-isolated, laboratory-confirmed, COVID-19 patients. Given the current absence of a vaccine against SARS-COV-2 and the lack of a specific treatment for -COVID-19, prevention strategies are of extreme importance. This project, designed to highly contribute to the international scientific debate on the role of avoidable lifestyle habits on COVID-19 severity, will provide valuable epidemiological data in order to support important recommendations to prevent COVID-19 incidence, progression and mortality.

Background Cannabis smoke affects the lungs similarly to tobacco smoke, causing symptoms such as increased cough, sputum, hyperinflation and chronic bronchitis. Chronic use can also cause serious lung diseases and airway obstruction. We developed and validated a method for the identification and quantification of cannabinol (CBN), cannabidiol (CBD), Δ-9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) in bronchoalveolar lavages (BALs) from hospitalized former or current tobacco smoking patients with lung disease and a long history of cannabis consumption and limited current tobacco use. Methods For the extraction of cannabinoids from BALs, a 1 mL sample was added with 300 µL of 0.1 N NaOH and 3 mL of hexane/ethyl acetate (9:1). The solvent was then evaporated to dryness. Trimethylsilyl derivatives were prepared and then analyzed by gas chromatography/mass spectrometry. Results The method was linear for the analytes under investigation with coefficients of determination of at least 0.99. Absolute analytical recovery was always better than 80%, imprecision and inaccuracy was always under 15%. Six cases out of 15 were positive for THC, CBN and CBD. In two BALs samples, the presence of 11-OH-THC was also measured while THC-COOH was not detected. In the six positive cases, the last cannabis smoking occurred in the previous 2-14 days. Conclusions This is the first time that cannabinoids have been detected in BALs, demonstrating the presence of a drug with its metabolites in a target organ of consumers who present with a lung disease. This occurrence let us hypothesize a role of cannabinoids in the development of the disease and prompted an investigation on possible associations between cannabis smoking and clinical outcomes in patients with lung disease and eventually evaluate a cytotoxic effect of cannabinoids themselves.

Phaeofungin (1), a new cyclic depsipeptide isolated from Phaeosphaeria sp., was discovered by application of reverse genetics technology, using the Candida albicans fitness test (CaFT). Phaeofungin is comprised of seven amino acids and a β,γ-dihydroxy-γ-methylhexadecanoic acid arranged in a 25-membered cyclic depsipeptide. Five of the amino acids were assigned with d-configurations. The structure was elucidated by 2D-NMR and HRMS-MS analysis of the natural product and its hydrolyzed linear peptide. The absolute configuration of the amino acids was determined by Marfey's method by complete and partial hydrolysis of 1. The CaFT profile of the phaeofungin-containing extract overlapped with that of phomafungin (3), another structurally different cyclic lipodepsipeptide isolated from a Phoma sp. using the same approach. Comparative biological characterization further demonstrated that these two fungal lipodepsipeptides are functionally distinct. While phomafungin was potentiated by cyclosporin A (an inhibitor of the calcineurin pathway), phaeofungin was synergized with aureobasidin A (2) (an inhibitor of the sphingolipid biosynthesis) and to some extent caspofungin (an inhibitor of glucan synthase). Furthermore, phaeofungin caused ATP release in wild-type C. albicans strains but phomafungin did not. It showed modest antifungal activity against C. albicans (MIC 16-32 μg/mL) and better activity against Aspergillus fumigatus (MIC 8-16 μg/mL) and Trichophyton mentagrophytes (MIC 4 μg/mL). The linear peptide was inactive, suggesting that the macrocyclic depsipeptide ring is essential for target engagement and antifungal activity.

Inhalers and nebulisers are devices used for delivering aerosolised drugs in subjects with Chronic Airflow Obstruction (CAO). This multicentre, cross-sectional observational study was performed in a large population of outpatients with CAO regularly using home aerosol therapy and referring to chest clinics. The aims of the study were to compare the characteristics of the group of subjects with CAO who were using home nebulisers but also experienced with inhalers vs. those only using inhalers and to investigate whether the first group of subjects was particularly prone to inhaler misuse. Information was gained evaluating the responses to a standardised questionnaire on home aerosol therapy and the observations of inhaler technique. We enrolled 1527 patients (58% males; mean ± SE; aged 61.1 ± 0.4 years; FEV1% pred 69.9 ± 0.6; 51% and 44% respectively suffering from COPD and asthma) who were only inhaler users (OIU group) and 137 (85% males; aged 67.7 ± 1.3 years; FEV1% pred 62.3 ± 2.9; 60% and 23% respectively suffering from COPD and asthma) who were using both nebulisers and inhalers (NIU group). Nebuliser users were older, had more severe obstruction, related symptoms and health care resources utilisation. Nebulisers users performed more critical inhalers errors than those of the OIU group (49% vs. 36%; p = 0.009). We conclude that our patients with CAO and regular nebuliser treatment had advanced age, severe respiratory conditions and common inhaler misuse., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We isolated a cyclic lipodepsipeptide, phomafungin, from a Phoma sp. The distinct antifungal activity of phomafungin in the crude extract was initially discovered by mechanistic profiling in the Candida albicans fitness test. The purified compound contains a 28 member ring consisting of eight amino acids and a beta-hydroxy-gamma-methyl-hexadecanoic acid, and displays a broad spectrum of antifungal activity against Candida spp., Aspergillus fumigatus and Trichophyton mentagrophytes with MIC of 2-8 microg/ml, and toxicity to mice at 25 mg/kg. The linear peptide derived from opening of the lactone ring was devoid of antifungal activity as well as toxicity. Phomafungin has been identified in a number of Phoma spp. collected from Africa and the Indian and Pacific Ocean islands.

A glycosylated tetramic acid, virgineone (1), was isolated from saprotrophic Lachnum virgineum. The antifungal activity of the fermentation extract of L. virgineum was characterized in the Candida albicans fitness test as distinguishable from other natural products tested. Bioassay-guided fractionation yielded 1, a tyrosine-derived tetramic acid with a C-22 oxygenated chain and a beta-mannose. It displayed broad-spectrum antifungal activity against Candida spp. and Aspergillus fumigatus with a MIC of 4 and 16 microg/mL, respectively. Virgineone was also identified in a number of Lachnum strains collected from diverse geographies and habitats.

Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.

Caspofungin inhibits synthesis of beta-D-1,3 glucan, essential to cell walls in Candida and Aspergillus spp., but activity against less common molds is largely uncharacterized. We demonstrate that caspofungin inhibits beta-D-1,3 glucan synthesis and reduces in vitro growth of clinical isolates from the genera Alternaria, Curvularia, Scedosporium, Acremonium, Bipolaris, and Trichoderma.

Aims: Natural fungal products were screened for antifungal compounds. The mode of action of one of the hits found and the taxonomy of the producing organism were analysed., Methods and Results: An extract from a Trichoderma species showed a more potent activity in an agar-based assay against the null mutant fks1::HIS strain than against the wild-type strain, suggesting that it could contain a glucan synthesis inhibitor. The active component was identified as the known compound ergokonin A. The compound exhibited activity against Candida and Aspergillus species, but was inactive against Cryptococcus species. It induced alterations in the hyphal morphology of Aspergillus fumigatus. The identification of the producing isolate was confirmed by sequencing of the rDNA internal transcribed spacers and comparison with the sequences of other Trichoderma species. The analysis showed that the producing fungus had a high homology with other strains classified as Trichoderma longibrachiatum and its teleomorph Hypocrea schweinitzii., Conclusions: The antifungal activity spectrum of ergokonin A and the morphology alterations induced on A. fumigatus are consistent with glucan synthesis as the target for ergokonin A. The production of ergokonin A is not uncommon, but is probably restricted to Trichoderma species., Significance and Impact of the Study: The discovery that ergokonin A could be an inhibitor of glucan synthesis, having a structure very different to other inhibitors, increases the likelihood that orally active agents with this fungal-specific mode of action may be developed.

Echinocandins, the lipopeptide class of glucan synthase inhibitors, are an alternative to ergosterol-synthesis inhibitors to treat candidiasis and aspergillosis. Their oral absorption, however, is low and they can only be used parenterally. During a natural product screening program for novel types of glucan synthesis inhibitors with improved bioavailability, a fungal extract was found that inhibited the growth of both a wild-type Saccharomyces cerevisiae strain and the null mutant of the FKS1 gene (fks1::HIS). The mutant strain was more sensitive to growth inhibition, suggesting that the fungal extract could contain an inhibitor of glucan synthesis. A novel acidic steroid, named arundifungin, was purified from a fungal extract obtained from a liquid culture of Arthrinium arundinis collected in Costa Rica. Arundifungin caused the same pattern of hallmark morphological alterations in Aspergillus fumigatus hyphae as echinocandins, further supporting the idea that arundifungin belongs to a new class of glucan synthesis inhibitors. Moreover, its antifungal spectrum was comparable to those of echinocandins and papulacandins, preferentially inhibiting the growth of Candida and Aspergillus strains, with very poor activity against Cryptococcus. Arundifungin was also detected in nine other fungal isolates which were ecologically and taxonomically unrelated, as assessed by sequencing of the ITS1 region. Further, it was also found in two more Arthrinium spp from tropical and temperate regions, in five psychrotolerant conspecific isolates collected on Macquarie Island (South Pacific) and belonging to the Leotiales, and in two endophytes collected in central Spain (a sterile fungus belonging to the Leotiales and an undetermined coelomycete).

In a screening of natural products with antifungal activity derived from endophytic fungi, we detected a potent activity in a culture belonging to the form-genus Hormonema, isolated from leaves of Juniperus communis. The compound is a new triterpene glycoside, showing an antifungal activity highly potent in vitro against Candida and Aspergillus and with moderate efficacy in an in vivo mouse model of disseminated candidiasis. The agent is especially interesting since its antifungal spectrum and its effect on morphology of Aspergillus fumigatus is comparable to that of the glucan synthase inhibitor pneumocandin B,,, the natural precursor of the clinical candidate MK-0991 (caspofungin acetate). An additional search for other Hormonema isolates producing improved titers or derivatives resulted in the isolation of two more strains recovered from the same plant host showing identical activity. The producing isolates were compared with other non-producing Hormonema strains by DNA fingerprinting and sequencing of the rDNA internal transcribed spacers. Comparison of rDNA sequences with other fungal species suggests that the producing fungus could be an undetermined Kabatina species. Kabatina is a coelomycetous genus whose members are known to produce Hormonema-like states in culture.

The incidence of bronchiectasis (BCT) has probably decreased in developed countries in recent years, but reliable statistical data on its occurrence are still lacking. The aim of the present study was to retrospectively evaluate the prevalence, age distribution and aetiology of BCT, diagnosed in a selected series of symptomatic patients of a Western country by using bronchography. The authors analysed the main known predisposing and associated conditions (PACs), and the occurrence and age distribution of BCT in 144 consecutive patients who underwent bronchological examination (fibreoptic bronchoscopy and bronchography) in the years 1987-1994 because of recurrent purulent bronchitis and/or haemoptysis. The overall prevalence of BCT was 34% (49/144); its age distribution was: 17.2% (0-10 yrs), 43.7% (11-20 yrs), 38% (21-30 yrs), 37.5% (31-40 yrs), 33.3% (41-50 yrs), 40% (51-60 yrs), and 20% (61-70 yrs). Thirty-one PACs were found in 29/144 patients of the whole study group. The prevalence of BCT was significantly higher in the subgroup of 29 patients with PACs than in the subgroup of 115 patients without PACs (75.9% versus 23.5%; p < 0000001). The aetiology of BCT was mainly unexplained, as it was only possible to detect 24 PACs in 22/49 patients with BCT (44.9%): congenital, genetic and immune disorders (eight), localized airways obstructive diseases (five), pulmonary infections (three), bronchial asthma (two), pulmonary lobar fibrosis (two), ulcerative colitis (two), dermatomiositis (one), and toxin inhalation (one). The authors conclude that bronchiectasis still occurs in a large percentage of symptomatic patients of a developed country in the post-antimicrobial era, especially in the second to sixth decades, as well as in the presence of predisposing and associated conditions; its aetiology remains unknown in more than half of cases.

We describe the case of a 57-yr-old female with endobronchial metastasis from stomach carcinoma. Respiratory symptoms began 3 months before the diagnosis of the gastric cancer. Chest computed tomography revealed a reticular lymphangitic carcinomatosis pattern with hilar and mediastinal lymphadenopathy. Fibreoptic bronchoscopy showed a spread submucosal infiltration which narrowed the apical segment of the left lower lobar bronchus. The biopsy specimen at that level was histologically identical to the gastric primitive cancer. To the authors' knowledge, stomach carcinoma has rarely been reported to give rise to airway metastases.

The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Natural-product inhibitors of cell wall (1,3)-beta-D-glucan synthase such as lipopeptide pneumocandins and echinocandins as well as the glycolipid papulacandins have been evaluated as potential therapeutics for the last two decades. As a result, MK-0991 (caspofungin acetate; Cancidas), a semisynthetic analogue of pneumocandin B(o), is being developed as a broad-spectrum parenteral agent for the treatment of aspergillosis and candidiasis. This and other lipopeptide antifungal agents have limited oral bioavailability. Thus, we have sought new chemical structures with the mode of action of lipopeptide antifungal agents but with the potential for oral absorption. Results of natural-product screening by a series of newly developed methods has led to the identification of four acidic terpenoid (1,3)-beta-D-glucan synthase inhibitors. Of the four compounds, the in vitro antifungal activity of one, enfumafungin, is comparable to that of L-733560, a close analogue of MK-0991. Like the lipopeptides, enfumafungin specifically inhibits glucan synthesis in whole cells and in (1,3)-beta-D-glucan synthase assays, alters the morphologies of yeasts and molds, and produces a unique response in Saccharomyces cerevisiae strains with point mutations in FKS1, the gene which encodes the large subunit of glucan synthase.

Coprophilin, a decalin pentanedienoic acid methyl ester, was isolated from an unidentified fungus by bioassay guided separation. It inhibited (MIC = 1.5 microM) the growth of Eimeria tenella in an in vitro assay. The isolation, structure elucidation, absolute stereochemistry and biology are described.

Cytosporin A, B and C, three antagonists of [125I]-angiotensin II binding to rat adrenal glands were discovered in fermentations of an endophytic Cytospora sp. during routine screening using semi-automated procedures. The most potent of these displayed an IC50 of 1.5-3 microM and was specific for angiotensin II AT2.

A novel oleic acid ester of the carotane sesquiterpene 14-hydroxy CAF-603 was isolated from Trichoderma virens grown in a solid brown rice-based medium, a solid millet-based medium, or a mannitol-based liquid medium. Its structure was determined on the basis of ms and nmr analysis. It retains distinct biological activity on the high conductance calcium-activated potassium channel, unlike its analogues 14-hydroxy CAF-603, CAF-603 3-oleate, or CAF-603 3-linoleate.

Potent antifungal activity was detected in fermentation extracts of Sporormiella australis and two related components were isolated from solid fermentations using silica gel and high speed countercurrent chromatography. The most active antifungal component, australifungin, contained a unique combination of alpha-diketone and beta-ketoaldehyde functional groups. Australifungin exhibited broad spectrum antifungal activity against human pathogenic fungi with MICs against Candida spp., Cryptococcus neoformans, and Aspergillus spp. between 0.015 and 1.0 microgram/ml. Mode of action studies revealed that australifungin interfered with fungal lipid metabolism by specifically inhibiting sphingolipid synthesis at the step converting sphinganine to ceramide.

We have developed a liquid fermentation medium for the submerged culture of the fungus, Zalerion arboricola, which supports the rapid production of an echinocandin-type antibiotic, pneumocandin A0 (formerly L-671,329), in yields increased at least 4-fold over those reported previously. The improvements were achieved through medium simplification, substitution of high levels of mannitol for glycerol as the major source of carbon, and restriction of available magnesium. Antibiotic formation in batch cultures with this mannitol-based medium is not confined to the idiophase; rather production appears to be biphasic, with synthesis beginning during growth (i.e., at day 3) and increasing in rate at day 11, well after rapid growth has ended. Accumulation of antibiotic continues beyond 14 days, and by 21 days titers greater than 500 micrograms/ml are attained. For the synthesis of a related compound, pneumocandin B0, by a mutant strain of Z. arboricola, the medium gives similar production kinetics and a titer of 800 micrograms/ml. Although supplementation of the medium with magnesium ions stimulates growth, it decreases titer by preferentially affecting the second phase of antibiotic synthesis. This decline in synthesis in the magnesium-supplemented medium is explained by the depletion of mannitol before the second phase of synthesis can begin. In contrast, mannitol in the magnesium-limited medium is used more slowly with approximately half still available at day 11 to support continued antibiotic formation.

In screening for antifungal inhibitors from fungi, four related antifungal agents have been isolated from the cultivation of Aspergillus fumigatus ATCC 20857. These agents were initially produced by the microorganism growing on a solid millet-based medium. A liquid medium containing both glucose and glycerol has also been developed in which these antibiotics are produced in two phases. These novel compounds, sphingofungins A, B, C, and D, show a limited spectrum of antifungal activity but were especially effective against Cryptococcus species.

A novel cytochalasin, L-696,474, (18-dehydroxy cytochalasin H) that inhibits HIV-1 protease was discovered in fermentations of a bark-inhabiting Ascomycete, Hypoxylon fragiforme. The product was first identified from extracts of an agar medium. Fermentation studies on a number of media indicated that the product can be made on several solid and liquid media. Optimum production was obtained from growth in a complex medium composed of glycerol, glucose, citrate, Ardamine, soybean meal, tomato paste, and inorganic salts. Other Hypoxylon spp., related species of Xylariales, and other fungi known to produce cytochalasins, were also surveyed for their ability to make L-696,474. Only one other Hypoxylon fragiforme isolate was found to make this novel cytochalasin; none of the other cultures surveyed made L-696,474 or any other compounds which inhibit HIV-1 protease.

In screening for new antifungal agents from fungi, a new lipopeptide antifungal agent, L-671,329, similar to echinocandin B, has been isolated from Zalerion arboricola. Studies indicate that L-671,329 is produced under both solid and liquid fermentation conditions.