Search

PURPOSE OF REVIEW: Environmental fungi such as Cryptococcus neoformans and Aspergillus fumigatus must survive many different and changing environments as they transition from their environmental niches to human lungs and other organs. Fungi alter their cell surfaces and secreted macromolecules to respond to and manipulate their surroundings. RECENT FINDINGS: This review focuses on exo-polysaccharides, chains of sugars that transported out of the cell and spread to the local environment. Major exo-polysaccharides for C. neoformans and A. fumigatus are glucuronylxylomannan (GXM) and galactosaminogalactan (GAG), respectively, which accumulate at high concentrations in growth medium and infected patients SUMMARY: Here we discuss GXM and GAG synthesis and export, their immunomodulatory properties, and their roles in biofilm formation. We also propose areas of future research to address outstanding questions in the field that could facilitate development of new disease treatments.

SUMMARYEnvironmental pathogens, which move from ecological niches to mammalian hosts, must adapt to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. When C. neoformans enters the lungs, infecting cells (30 μm diameter), then form a heterogeneous population. The brain contains uniformly small cells (∼7 μm). We demonstrate that formation of a small C. neoformans morphotype – called “seed” cells due to their disseminating ability – is critical for extrapulmonary organ entry. Seed cell formation is triggered by environmental factors, including C. neoformans’ environmental niche, pigeon guano. The underlying trigger, phosphate, can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. We demonstrate that C. neoformans’ size variation is not just a continuum but inducible subpopulations that change host interactions to facilitate microbe survival and spread.

Invasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited treatments. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits growth instead of killing fungal cells, necessitating long treatments. To improve treatment, we used our novel high-throughput method, the overlap2 method (O2M) to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole. In vivo, this combination doubled the time-to-endpoint of mice with Cryptococcus neoformans meningitis. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes., eLife digest Individuals with weakened immune systems – such as recipients of organ transplants – can fall prey to illnesses caused by fungi that are harmless to most people. These infections are difficult to manage because few treatments exist to fight fungi, and many have severe side effects. Antifungal drugs usually slow the growth of fungi cells rather than kill them, which means that patients must remain under treatment for a long time, or even for life. One way to boost efficiency and combat resistant infections is to combine antifungal treatments with drugs that work in complementary ways: the drugs strengthen each other’s actions, and together they can potentially kill the fungus rather than slow its progression. However, not all drug combinations are helpful. In fact, certain drugs may interact in ways that make treatment less effective. This is particularly concerning because people with weakened immune systems often take many types of medications. Here, Wambaugh et al. harnessed a new high-throughput system to screen how 2,000 drugs (many of which already approved to treat other conditions) affected the efficiency of a common antifungal called fluconazole. This highlighted 19 drugs that made fluconazole less effective, some being antibiotics routinely used to treat patients with weakened immune systems. On the other hand, 40 drugs boosted the efficiency of fluconazole, including dicyclomine, a compound currently used to treat inflammatory bowel syndrome. In fact, pairing dicyclomine and fluconazole more than doubled the survival rate of mice with severe fungal infections. The combined treatment could target many species of harmful fungi, even those that had become resistant to fluconazole alone. The results by Wambaugh et al. point towards better treatments for individuals with serious fungal infections. Drugs already in circulation for other conditions could be used to boost the efficiency of fluconazole, while antibiotics that do not decrease the efficiency of this medication should be selected to treat at-risk patients.

SummaryInvasive fungal infections cause 1.6 million deaths annually, primarily in immunocompromised individuals. Mortality rates are as high as 90% due to limited number of efficacious drugs and poor drug availability. The azole class antifungal, fluconazole, is widely available and has multi-species activity but only inhibits fungal cell growth instead of killing fungal cells, necessitating long treatments. To improve fluconazole treatments, we used our novel high-throughput method, the overlap2method (O2M), to identify drugs that interact with fluconazole, either increasing or decreasing efficacy. Although serendipitous identification of these interactions is rare, O2M allows us to screen molecules five times faster than testing combinations individually and greatly enriches for interactors. We identified 40 molecules that act synergistically (amplify activity) and 19 molecules that act antagonistically (decrease efficacy) when combined with fluconazole. We found that critical frontline beta-lactam antibiotics antagonize fluconazole activity. A promising fluconazole-synergizing anticholinergic drug, dicyclomine, increases fungal cell permeability and inhibits nutrient intake when combined with fluconazole.In vivo, this combination doubled the time-to-endpoint of mice with disseminatedCryptococcus neoformansinfections. Thus, our ability to rapidly identify synergistic and antagonistic drug interactions can potentially alter the patient outcomes.

Intergranular interactions and thermal stability factors in Fe-Ti-N thin films were studied as a function of nitrogen content. This was done through: 1) measurements of the isothermal remanence magnetization and direct current demagnetization, coupled with an analysis based on a modified Che-Bertram interaction field model for the intergranular interactions and 2) measurements of the dependence of the coercive force on the holding time for the applied field, coupled with a Sharrock analysis in the case of the thermal stability. The data indicate that the intergranular interactions are mainly exchange dominated for nitrogen levels in the 0-4 at.% range, become primarily magnetostatic for 4-7 at.% nitrogen, and switch back to exchange dominated for higher levels. For nitrogen amounts in the 7-11 at.% range, the thermal stability factors are largest at about 7 at.% and decrease for higher nitrogen amounts. The inferred unit switching lateral size from the Sharrock analysis is about 10-20 times larger than the grain size. The changes in the intergranular interactions and the thermal stability factors with nitrogen content can be qualitatively explained by a grain size effect.

The commensal microbiota has an important impact on host health, which is only beginning to be elucidated. Despite the presence of fungal, archaeal, and viral members, most studies have focused solely on the bacterial microbiota. Antibodies against the yeast Saccharomyces cerevisiae are found in some patients with Crohn’s disease (CD), suggesting that the mycobiota may contribute to disease severity. We report that S. cerevisiae exacerbated intestinal disease in a mouse model of colitis and increased gut barrier permeability. Transcriptome analysis of colon tissue from germ-free mice inoculated with S. cerevisiae or another fungus, Rhodotorula aurantiaca, revealed that S. cerevisiae colonization affected the intestinal barrier and host metabolism. A fecal metabolomics screen of germ-free animals demonstrated that S. cerevisiae colonization enhanced host purine metabolism, leading to an increase in uric acid production. Treatment with uric acid alone worsened disease and increased gut permeability. Allopurinol, a clinical drug used to reduce uric acid, ameliorated colitis induced by S. cerevisiae in mice. In addition, we found a positive correlation between elevated uric acid and anti-yeast antibodies in human sera. Thus, yeast in the gut may be able to potentiate metabolite production that negatively affects the course of inflammatory bowel disease.

Antibiotic-resistant infections kill approximately 23,000 people and cost $20,000,000,000 each year in the United States alone despite the widespread use of small-molecule antimicrobial combination therapy. Antibiotic combinations typically have an additive effect: the efficacy of the combination matches the sum of the efficacies of each antibiotic when used alone. Small molecules can also act synergistically when the efficacy of the combination is greater than the additive efficacy. However, synergistic combinations are rare and have been historically difficult to identify. High-throughput identification of synergistic pairs is limited by the scale of potential combinations: a modest collection of 1,000 small molecules involves 1 million pairwise combinations. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, the overlap2 method (O2M). O2M extracts patterns from chemical-genetic datasets, which are created when a collection of mutants is grown in the presence of hundreds of different small molecules, producing a precise set of phenotypes induced by each small molecule across the mutant set. The identification of mutants that show the same phenotype when treated with known synergistic molecules allows us to pinpoint additional molecule combinations that also act synergistically. As a proof of concept, we focus on combinations with the antibiotics trimethoprim and sulfamethizole, which had been standard treatment against urinary tract infections until widespread resistance decreased efficacy. Using O2M, we screened a library of 2,000 small molecules and identified several that synergize with the antibiotic trimethoprim and/or sulfamethizole. The most potent of these synergistic interactions is with the antiviral drug azidothymidine (AZT). We then demonstrate that understanding the molecular mechanism underlying small-molecule synergistic interactions allows the rational design of additional combinations that bypass drug resistance. Trimethoprim and sulfamethizole are both folate biosynthesis inhibitors. We find that this activity disrupts nucleotide homeostasis, which blocks DNA replication in the presence of AZT. Building on these data, we show that other small molecules that disrupt nucleotide homeostasis through other mechanisms (hydroxyurea and floxuridine) also act synergistically with AZT. These novel combinations inhibit the growth and virulence of trimethoprim-resistant clinical Escherichia coli and Klebsiella pneumoniae isolates, suggesting that they may be able to be rapidly advanced into clinical use. In sum, we present a generalizable method to screen for novel synergistic combinations, to identify particular mechanisms resulting in synergy, and to use the mechanistic knowledge to rationally design new combinations that bypass drug resistance., Author summary Antibiotic resistance is a growing problem that threatens our ability to treat systemic bacterial infections. One strategy to combat antibiotic resistance is the use of synergistic antibiotic pairs that, when combined, have activity that is considerably greater than the sum of each individual drug’s activity on its own. Synergistic combinations can even inhibit the growth of bacteria that are resistant to the individual treatment drugs. However, synergistic pairs are rare and difficult to identify. High-throughput identification of synergistic pairs is challenging due to scale: 1 million different pairs are possible for a relatively small collection of 1,000 small molecules. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, termed the overlap2 method (O2M), that dramatically speeds up the screening process. First, we identify mutants that show the same phenotype when treated with each individual molecule in a synergistic pair, then use this information to guide screens for additional synergistic pairs. As a proof of concept, we studied the synergistic antibiotic pair trimethoprim and sulfamethizole, and we identified several additional synergistic molecules. Among these is the antiviral drug azidothymidine (AZT), which blocks bacterial DNA replication. Trimethoprim and sulfamethizole both inhibit folate biosynthesis, which is necessary for the proper synthesis of nucleotides for DNA replication and repair. We found that reduced nucleotide levels sensitize E. coli cells to AZT. When we substitute trimethoprim with other small molecules that also reduce nucleotide levels, we find that these small molecules also act synergistically with AZT. Indeed, AZT in combination with trimethoprim substitutes inhibits the growth of trimethoprim-resistant clinical isolates more potently than trimethoprim and AZT or trimethoprim and sulfamethizole. This work demonstrates that when we resolve the pathways that underlie synergistic interactions, we can then identify additional small molecules that act by similar mechanisms, providing a means to bypass antibiotic resistance.

The aflatoxin biosynthetic pathway represents one of the est studied pathways of fungal secondary metabolism. Its lucidation is the result of over 30 years of study by cientists in many disciplines. For recent reviews on the hemistry of the pathway see articles by Bhatnagar et al. 1992) Minto and Townsend (1997), and Woloshuk and rieto (1997). Concern over the toxicity and carcinogenicty of aflatoxin has been the prime force driving research in his area. Aflatoxin B1 (AFB1) is the most potent naturally ccurring carcinogen known (Squire, 1989), and epidemioogical data implicate aflatoxin as a component of liver ancer in humans in certain parts of the world (Hall and ild, 1994). Although aflatoxins are not extremely toxic, onsumption of aflatoxin contaminated food by animals can ead to decreased weight gain, hemorrhaging, and suppression f the immune system (Miller and Wilson, 1994). Aflatoxins are produced by four species of Aspergillus: . flavus, A. parasiticus, A. nomius, and A. tamarii (Payne, 998; Kurtzman et al., 1986; Goto et al., 1996). From an gricultural standpoint, the two most significant fungi are

Four bacteriophages (phages) growing on the same Leuconostoc strain were characterised. Electron micrographs showed these phages to be similar in morphology to the commonly isolated lactococcal phages with head diameters ranging from 49–55 nm and tail lengths of 117–131 nm. A distinctive base plate and collar were also present. From restriction enzyme analysis of purified phage DNA, the genome sizes were 23–29 kb. All four phages showed one major structural protein (of approximately 24 kDa) on SDS polyacrylamide gels. Hybridization experiments confirmed that the phages belonged to the same homology group. There was no homology between DNA from these phages and DNA from a prolate or small isometric lactococcal phage.

This paper reports on the application of the ligase chain reaction (LCR) to the specific detection of variants of the nisin structural gene (nisinA and nisinZ) in nisin producing strains of Lactococcus lactis ssp lactis. The LCR assay was used to screen nisin producing strains to determine which form of the nisin structural gene they contained. This method of differentiating the nisin structural gene variants provides a useful alternative to the only other available genetic differentiation, that of sequencing the gene.

The Durham route to polyacetylene provides, for the first time, the possibility of controlling the crystalline structure of this fascinating material. This control will enable us to probe the role of the redox doping and relative spatial co-ordinates of the chains in determining electrical conductivity. Before this can be accomplished it is necessary to understand the nature of the order within the variety of materials which can now be produced. Material produced from quiescent films can have a range of structures from largely amorphous (inasmuch as a structure composed of rigid rods can exhibit disorder) to slightly crystalline. By stretching the precursor film during transformation highly oriented, crystalline polyacetylene can be produced. This offers a range of structures and should enable the separation of inter- and intra-chain electronic effects for the first time.

The structure of polyacetylene produced from a precursor polymer is compared with that of polymer produced by a soluble Ziegler-Natta catalyst. X-ray diffraction shows that the precursor polyacetylene can be produced with a wide range of ordering from apparently amorphous to crystalline, although the ordering is never as high as that of polymer produced by the Ziegler-Natta route. An increase in ordering, as measured by X-ray peak width, has been observed during isomerization; this contrasts with normal polyacetylene where no change in order is found. Both forms of polyacetylene show a gradual shift in the interchain d -spacing from 395 pm ( cis/trans mixed) to 365 pm ( trans ). Since only a single peak is found in this region the cis - and trans -forms are not phase-separated on a scale of 3 nm or more. Small-angle X-ray scattering also indicates that discrete crystalline and amorphous regions, as found in polyethylene, are not present

The processes involved in the thermal transformation of ‘Durham polyacetylene precursor’, poly[[5,6-bis(trifluoromethyl)-bicyclo[2,2,2]octa-5,7-diene-2,3-diyl]-1,2-ethenediyl], to all- trans polyacetylene have been studied. Above about 100°C the reactions are virtually concurrent, but at 50°C one can distinguish a first-order elimination ( k = 7.6 × 10 −4 s −1 ), effusion of the elimination product ( D = 4 × 10 −9 cm 2 s −1 ), and a first order cis-trans isomerization ( k = 1.5 × 10 −5 s −1 ). The isomerization is very sensitive to traces of oxygen below 80°C, but at higher temperatures this is less pronounced and the reaction becomes approximately second order. Isomerization is generally more facile in the less ordered Durham polymer than in polymers from the Shirakawa route.

Mechanistic aspects of the two step Durham route to polyacetylene are discussed. It is shown that a catalyst based on MoCl5 gives a higher proportion of cis vinylenes in both precursor polymer and nascent polyacetylene than does a WC16 based system.

Polyacetylene prepared by the Shirakawa route is a mat of crystalline fibrills, which are capable of being oxidized or reduced to a highly conducting state. Polyacetylene can also be produced from a soluble precursor polymer by a thermally driven transformation reaction (the ‘Durham’ route) as a solid, coherent film. This transformation consists of the elimination of a substituted benzene unit followed by the isomerization of the largely cis polyacetylene produced to a disordered trans isomer. We have studied the effect of different transformation conditions and from these studies it is possible to gain an insight into these processes. Techniques such as caloremetry, infrared, resonance Raman and optical spectroscopy, e.s.r., X-ray diffraction and electrical conductivity have all been used to probe the transformation and the product. The resultant information gives a consistent picture of the overall transformation processes detailed above but also shows an ordering associated with higher transformation temperatures. A thorough knowledge of these processes enables the orientation of the precursor to produce highly crystalline material.

An investigation has been carried out aimed at developing a process for the growth of large crystals of synthetic quartz using silica-rich rocks native to the United Kingdom as the raw material. As a result of this work the effect of impurities on the habit and quality of the crystals has been determined. The role of aluminium as a key impurity has been proved and the darkening produced by X-irradiation has been used to study the distribution of impurities in the synthetic quartz. Recent work has thrown further light on the manner in which aluminium, possibly associated with another element, is incorporated in the quartz structure.

ATP-depletion of red blood cells (RBCs) by glucose deprivation transforms RBCs from the normal discocyte configuration into echinocytes. In the course of this shape change, there is a significant loss of membrane sialic acid, a decrease in cell surface negativity, and survival studies show that RBCs have an abbreviated life span. It is not known if the loss of negativity and decreased survival are directly related to nucleotide depletion or if they may be an inevitable consequence of the discocyte-echinocyte shape transformation.In the current study, we examined the effects of rapid discocyte-echinocyte transformations on RBC surface charge, as measured by cationized ferritin binding and agglutination rates in the presence of poly-L-lysine. Lysolecithin (LYL) is a strongly echinocytogenic lipid that transforms human RBCs into echinocytes in > 5 minutes, in contrast to ATP depletion by the method of Weed et al, which requires many hours to accomplish the same transformation.