Drug Leads from Endophytic Fungi: Lessons Learned via Scaled Production.

Recently, the isolation and elucidation of a series of polyhydroxyanthraquinones were reported from an organic extract of a solid phase culture of an endophytic fungus, Penicillium restrictum (strain G85). One of these compounds, ω-hydroxyemodin (1: ), showed promising quorum-sensing inhibition against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) in both in vitro and in vivo models. The initial supply of 1: was 19 mg, and this amount needed to be scaled by a factor of 30 to 50 times, in order to generate material for further in vivo studies. To do so, improvements were implemented to enhance both the fermentation of the fungal culture and the isolation of this compound, with the target of generating > 800 mg of study materials in a period of 13 wk. Valuable insights, both regarding chemistry and mycology, were gained during the targeted production of 1: on the laboratory-scale. In addition, methods were modified to make the process more environmentally friendly by judicious choice of solvents, implementing procedures for solvent recycling, and minimizing the use of halogenated solvents.

New tricks for old dogs: Two new macrocyclic trichothecene epimers and absolute configuration of 16-hydroxyverrucarin B.

Two new compounds, 3'-epi-16-hydroxyverrucarin A and 3'-epiverrucarin X, have been isolated and identified, and the characterization data of a series of known trichothecenes have been refined. The interesting structure and potent biological activities of macrocyclic trichothecenes have been of interest to the scientific community for several decades. However, some of the characterization data for the older analogues of this class are not well documented, either because of a lack of absolute configuration or a lack of clarity in the NMR data, largely due to technological limitations at the time they were discovered. NMR techniques, application of Mosher's esters analysis, and electronic circular dichroism were used here both to refine the characterization of known trichothecenes, as well as to uncover new structures. These studies demonstrate strategies that can be used to interrogate the characterization data of well-known secondary metabolites, thereby gaining greater insight into methods that can be used to refine previous literature.

The association of material deprivation component measures with injury hospital separations in British Columbia, Canada.

BACKGROUND: This study examines social disparities across neighbourhood levels of income, education and employment in relation to overall injury hospital separations in the province of British Columbia, Canada. Further, the study examines the relationships of social disparities to a set of three injury prevention priorities in British Columbia, namely, transport (motor vehicle occupant, pedestrian and cyclist), falls among older adults, and youth self-harm. The goal being to better understand area-based injury incidence with a view to precision prevention initiatives, particularly for more vulnerable populations. METHODS: Acute hospital separations from the Discharge Abstract Database were identified for all causes of injury and the three BC injury prevention priorities for the period April 1, 2009 to March 31, 2014, inclusive. An ecological approach was applied where each hospital separation case was attributed with the income, education and employment level according to the injured individual's area of residence, derived from the 2011 CensusPlus data. RESULTS: Injury hospital separation data were available for 191 Forward Sortation Areas in BC. Between April 1, 2009 and March 31, 2014, there was a total of 177,861 injury-related hospital separations, averaging 35,572 hospital separations per year and an annual rate of 779 injury hospital separations per 100,000 population. Injury hospital separation rates varied with the measured neighbourhood area socioeconomic status variables. Injury hospital separation rates demonstrated an inverse relationship with neighbourhood levels of income and education. Neighbourhood area socioeconomic status differences were also associated with the injury hospital separation rates for falls among older adults, motor vehicle crashes involving motor vehicle occupants, pedestrians, cyclists and young drivers, and youth self-harm. CONCLUSIONS: The study results show that neighbourhood levels of income, education and employment are associated with the risk of injury hospital separation. In particular, low education levels in FSAs was associated with increased risk of injury hospital separation, mainly for motor vehicle occupants, pedestrians, young drivers, and youth self-harm. The results of this study provide useful information for implementing injury prevention initiatives and interventions in BC to align with the provincial public health system and road safety strategy goals, particularly for identified priorities.

Droplet probe: coupling chromatography to the in situ evaluation of the chemistry of nature.

Covering: up to 2019The chemistry of nature can be beautiful, inspiring, beneficial and poisonous, depending on perspective. Since the isolation of the first secondary metabolites roughly two centuries ago, much of the chemical research on natural products has been both reductionist and static. Typically, compounds were isolated and characterized from the extract of an entire organism from a single time point. While there could be subtexts to that approach, the general premise has been to determine the chemistry with very little in the way of tools to differentiate spatial and/or temporal changes in secondary metabolite profiles. However, the past decade has seen exponential advances in our ability to observe, measure, and visualize the chemistry of nature in situ. Many of those techniques have been reviewed in this journal, and most are tapping into the power of mass spectrometry to analyze a plethora of sample types. In nearly all of the other techniques used to study chemistry in situ, the element of chromatography has been eliminated, instead using various ionization sources to coax ions of the secondary metabolites directly into the mass spectrometer as a mixture. Much of that science has been driven by the great advances in ambient ionization techniques used with a suite of mass spectrometry platforms, including the alphabet soup from DESI to LAESI to MALDI. This review discusses the one in situ analysis technique that incorporates chromatography, being the droplet-liquid microjunction-surface sampling probe, which is more easily termed "droplet probe". In addition to comparing and contrasting the droplet probe with other techniques, we provide perspective on why scientists, particularly those steeped in natural products chemistry training, may want to include chromatography in in situ analyses. Moreover, we provide justification for droplet sampling, especially for samples with delicate and/or non-uniform topographies. Furthermore, while the droplet probe has been used the most in the analysis of fungal cultures, we digest a variety of other applications, ranging from cyanobacteria, to plant parts, and even delicate documents, such as herbarium specimens.

Area-based socioeconomic disparities in mortality due to unintentional injury and youth suicide in British Columbia, 2009-2013. / Disparités socioéconomiques et spatiales dans les décès attribuables au suicide chez les jeunes et aux blessures non intentionnelles en Colombie-Britannique (2009-2013).

INTRODUCTION: The association between health outcomes and socioeconomic status (SES) has been widely documented, and mortality due to unintentional injuries continues to rank among the leading causes of death among British Columbians. This paper quantified the SES-related disparities in the mortality burden of three British Columbia's provincial injury prevention priority areas: falls among seniors, transport injury, and youth suicide. METHODS: Mortality data (2009 to 2013) from Vital Statistics and dissemination area or local health area level socioeconomic data from CensusPlus 2011 were linked to examine age-standardized mortality rates (ASMRs) and disparities in ASMRs of unintentional injuries and subtypes including falls among seniors (aged 65+) and transport-related injuries as well as the intentional injury type of youth suicide (aged 15 to 24). Disparities by sex and geography were examined, and relative and absolute disparities were calculated between the least and most privileged areas based on income, education, employment, material deprivation, and social deprivation quintiles. RESULTS: Our study highlighted significant sex differences in the mortality burden of falls among seniors, transport injury, and youth suicide with males experiencing significantly higher mortality rates. Notable geographic variations in overall unintentional injury ASMR were also observed across the province. In general, people living in areas with lower income and higher levels of material deprivation had increasingly higher mortality rates compared to their counterparts living in more privileged areas. CONCLUSION: The significant differences in unintentional and intentional injury-related mortality outcomes between the sexes and by SES present opportunities for targeted prevention strategies that address the disparities.

Calothrixamides A and B from the Cultured Cyanobacterium Calothrix sp. UIC 10520.

Cyanobacteria are a source of chemically diverse metabolites with potential medicinal and biotechnological applications. Rapid identification of compounds is central to expedite the natural product discovery process. Mass spectrometry has been shown to be an important tool for dereplication of complex natural product samples. In addition, chromatographic separation and complementary spectroscopic analysis (e.g., UV) can enhance the confidence of the dereplication process. Here, we applied a droplet-liquid microjunction-surface sampling probe (droplet probe) coupled with UPLC-PDA-HRMS-MS/MS to identify two new natural products in situ from the freshwater strain Calothrix sp. UIC 10520. This allowed us to prioritize this strain for chemical investigation based on the presence of new metabolites very early in our discovery process, saving both time and resources. Subsequently, calothrixamides A (1) and B (2) were isolated from large-scale cultures, and the structures were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configurations were determined by a combination of chemical degradation reactions, derivatization methods (Mosher's, Marfey's, and phenylglycine methyl ester), and J-based configurational analysis. Calothrixamides showed no cytotoxic activity against the MDA-MB-435, MDA-MB-231, and OVCAR3 cancer cell lines. They represent the first functionalized long-chain fatty acid amides reported from the Calothrix genus and from a freshwater cyanobacterium.

Non-Destructive Chemical Analysis of a Garcinia mangostana L. (Mangosteen) Herbarium Voucher Specimen.

Herbarium voucher specimens are used primarily for taxonomic confirmation. However, they also afford a record of the metabolic profile of a plant, potentially at the time it was collected, or at the very least, at the time of analysis. Even with the enhanced sensitivity of modern analytical techniques, analysis of the metabolites of a herbarium voucher requires removal and consumption of at least part of an entire specimen. We present herein a non-destructive method to analyze the metabolites of herbarium voucher specimens with the droplet-liquid microjunction-surface sampling probe (droplet probe) coupled to ultra-performance liquid chromatography and highresolution mass spectrometry. As proof of concept, a herbarium voucher specimen of Garcinia mangostana (mangosteen) was utilized due to the well-characterized xanthones biosynthesized by this plant, which are of interest as potential anticancer agents. Also, the juice of the fruits of this plant is used widely in the United States and in other countries as a botanical dietary supplement. Metabolite profiles of the sampled surfaces were compared to a subset of xanthone standards. Using this innovative method on the herbarium voucher specimen, we were able to readily identify cytotoxic prenylated xanthones while maintaining the integrity of the entire specimen.

Enhanced dereplication of fungal cultures via use of mass defect filtering.

Effective and rapid dereplication is a hallmark of present-day drug discovery from natural sources. This project strove to both decrease the time and expand the structural diversity associated with dereplication methodologies. A 5 min liquid chromatographic run time employing heated electrospray ionization (HESI) was evaluated to determine whether it could be used as a faster alternative over the 10 min ESI method we reported previously. Results revealed that the 5 min method was as sensitive as the 10 min method and, obviously, was twice as fast. To facilitate dereplication, the retention times, UV absorption maxima, full-scan HRMS and MS/MS were cross-referenced with an in-house database of over 300 fungal secondary metabolites. However, this strategy was dependent upon the makeup of the screening in-house database. Thus, mass defect filtering (MDF) was explored as an additional targeted screening strategy to permit identification of structurally related components. The use of a dereplication platform incorporating the 5 min chromatographic method together with MDF facilitated rapid and effective identification of known compounds and detection of structurally related analogs in extracts of fungal cultures.

Secondary Metabolites from Fungal Endophytes of Echinacea purpurea Suppress Cytokine Secretion by Macrophage-Type Cells.

Botanical extracts of Echinacea purpurea have been widely used for the treatment of upper respiratory infections. We sought to chemically examine fungal endophytes inhabiting E. purpurea, and to identify compounds produced by these endophytes with in vitro cytokine-suppressive activity. Twelve isolates from surface sterilized seeds of E. purpurea were subjected to fractionation and major components were isolated. Sixteen secondary metabolites belonging to different structural classes were identified from these isolates based on NMR and mass spectrometry data. The compounds were tested for their influence on cytokine secretion by murine macrophage-type cells. Alternariol (1), O-prenylporriolide (4), porritoxin (10) ß-zearalenol (13), and (S)-zearalenone (14) inhibited production of TNF-α from RAW 264.7 macrophages stimulated with LPS in the absence of any significant cytotoxicity. This is the first report of a cytokine-suppressive effect for 4. The results of this study are particularly interesting given that they show the presence of compounds with cytokine-suppressive activity in endophytes from a botanical used to treat inflammation. Future investigations into the role of fungal endophytes in the biological activity of E. purpurea dietary supplements may be warranted.

Effect of diabetes on tuberculosis control in 13 countries with high tuberculosis: a modelling study.

BACKGROUND: Diabetes increases the risk of tuberculosis incidence and the risk of adverse treatment outcomes in patients with tuberculosis. Because prevalence of diabetes is increasing in low-income and middle-income countries where the burden of tuberculosis is high, prevention of diabetes carries the potential to improve tuberculosis control worldwide. METHODS: We used dynamic tuberculosis transmission models to analyse the potential effect of diabetes on tuberculosis epidemiology in 13 countries with high tuberculosis burden. We used data for previous diabetes prevalence in each country and constructed scenarios to represent the potential ranges of future diabetes prevalence. The country-specific model was calibrated to the estimated trend of tuberculosis incidence. We estimated the tuberculosis burden that can be reduced by alternative scenarios of diabetes prevention. FINDINGS: If the prevalence of diabetes continues to rise as it has been in the past decade in the 13 countries (base case scenario), by 2035, the cumulative reduction in tuberculosis incidence would be 8·8% (95% credible interval [CrI] 4·0-15·8) and mortality would be 34·0% (30·3-39·6). Lowering the prevalence of diabetes by an absolute level of 6·6-13·8% could accelerate the decline of tuberculosis incidence by an absolute level of 11·5-25·2% and tuberculosis mortality by 8·7-19·4%. Compared with the base case scenario, stopping the rise of diabetes would avoid 6·0 million (95% CrI 5·1-6·9) incident cases and 1·1 million (1·0-1·3) tuberculosis deaths in 13 countries during 20 years. If interventions reduce diabetes incidence by 35% by 2025, 7·8 million (6·7-9·0) tuberculosis cases and 1·5 million (1·3-1·7) tuberculosis deaths could be averted by 2035. INTERPRETATION: The diabetes epidemic could substantially affect tuberculosis epidemiology in high burden countries. The communicable disease and non-communicable disease sectors need to move beyond conventional boundaries and link with each other to form a joint response to diabetes and tuberculosis. FUNDING: Taiwan National Science Council.

Quality of life and needs assessment on people living with HIV and AIDS in Malawi.

PURPOSE: Malawi is a sub-Saharan African nation with a severe HIV epidemic. The quality of life (QoL) has never been investigated among people living with HIV and AIDS (PLWHA) in Malawi. This study examines the QoL and associated factors including life needs among PLWHA at different stages of their illness in the northern region of Malawi. METHODS: Survey analysis of consecutive outpatient participants receiving highly active antiretroviral therapy at the Rainbow Clinic and non-HIV patients receiving care at the affiliated Mzuzu Central Hospital during a one-month period was performed. Laboratory testing and clinical diagnosis were used to determine HIV status, determine CD4 count, and classify WHO clinical stage. A total of 267 HIV-infected patients and 598 non-HIV participants completed a needs assessment and a Short Form-36 (SF-36) questionnaire, which contained a QoL subscale. SF-36 subscales and needs assessment scores were analyzed using t-test, ANOVA test, and Generalized Linear Model-Tukey's test. RESULTS: HIV-positive patients had significantly lower physical functioning (p=0.0365), mental health (p=0.001), social functioning (p<0.0001), and mental component summary (p=0.0069) scores than HIV-negative patients. Further, WHO Stage III HIV patients had significantly lower vitality (p=0.0439) and mental health (p=0.0022) scores than WHO Stages I and II patients; and WHO Stage IV patients had significantly lower vitality (p=0.0015), mental health (p=0.0006), and physical component summary (p=0.0443) scores than WHO Stages I and II patients. Finally, AIDS patients, as determined by CD4 count, had significantly lower bodily pain (p=0.0423) and physical component summary (p=0.0148) scores than non-AIDS, HIV-positive patients. CONCLUSION: HIV patients undergoing treatment in Malawi have a significantly lower QoL, both mentally and physically, than their non-HIV counterparts. Further, HIV patients at more advanced stages, both by the WHO definition and by CD4 count, have a significantly lower QoL than HIV patients at earlier stages of the disease.

Molecular epidemiology of HIV-1 subtype B, CRF01_AE, and CRF07_BC infection among injection drug users in Taiwan.

OBJECTIVE: An explosive outbreak of HIV-1 circulating recombinant form (CRF) 07_BC among injection drug users (IDUs) in Taiwan was first reported in 2004 and reach the peak in 2005. The objectives of this study were to investigate the molecular epidemiology of different HIV-1 subtypes and their associated risk factors among Taiwanese IDUs in 2004 and 2005. METHODS: Questionnaires and blood specimens were collected from inmates from 4 detention centers and 2 prisons. HIV-1 subtypes were determined using nested polymerase chain reactions with multiplex primers and phylogenetic analyses. A case-control study was conducted to elucidate risk factors associated with CRF07_BC infection. RESULTS: A total of 93.8%, 4%, and 2.2% of 451 inmates with IDU history were infected with CRF07_BC, subtype B, and CRF01_AE, respectively. Besides CRF07_BC, a new outbreak of CRF01_AE infection was identified among IDUs from central region. Multivariate analysis showed that sharing dissolved heroine solution [odds ratio (OR) = 17.2], sharing syringes (OR = 34), number of persons sharing syringes (2 vs. 1, OR = 3.1), and lower educational level (OR = 2.3) were risk factors associated with CRF07_BC infection. CONCLUSIONS: Sharing dissolved heroine solution is a neglected risk factor associated with HIV-1 infection and it should be emphasized in the AIDS education and harm reduction programs immediately.

Molecular evidence for the monophyly of tenrecidae (mammalia) and the timing of the colonization of Madagascar by Malagasy Tenrecs.

Tenrecs are a diverse family of insectivores, with an Afro-Malagasian biogeographic distribution. Three subfamilies (Geogalinae, Oryzorictinae, Tenrecinae) are restricted to Madagascar and one subfamily, the otter shrews (Potamogalinae), occurs on the mainland. Morphological studies have generated conflicting hypotheses according to which both tenrecids and Malagassy tenrecs are either monophyletic or paraphyletic. Competing hypotheses have different implications for the biogeographic history of Tenrecidae. At present, there are no molecular studies that address these hypotheses. The present study provides sequences of a nuclear protein-coding gene (vWF) and the mitochondrial 12S rRNA, tRNA valine, and 16S rRNA genes from a potamogaline (Micropotamogale). New sequences of these genes are also reported for the tenrecine, Tenrec ecaudatus. The 12S sequences from these taxa were combined with data already available for this locus from two other tenrecids (Echinops telfairi, subfamily Tenrecinae and Oryzorictes talpoides, subfamily Oryzorictinae). Phylogenetic analyses provided strong bootstrap support for the monophyly of Tenrecidae and Malagasy tenrecs. The majority of statistical tests rejected morphological claims for both a Tenrecinae--Chrysochloridae clade and an Oryzorictinae--Potamogalinae clade. Molecular clock estimates suggest a split of otter shrews and Malagasy tenrecs at approximately 53 MYA. We estimate that the ancestor of Malagasy tenrecs dispersed to Madagascar subsequent to this split but prior to about 37 MYA.