Monovalent Copper Ions Inhibit Enzymatic Systems

The effect of monovalent copper ions on enzymatic systems has hardly been studied to date; this is due to the low stability of monovalent copper ions in aqueous solutions, which led to the assumption that their concentration is negligible in biological systems. However, in an anaerobic atmosphere, and in the presence of a ligand that stabilizes the monovalent copper ions over the divalent copper ions, high and stable concentrations of monovalent copper ions can be reached. Moreover, the cell cytoplasm has a substantial concentration of potential stabilizers that can explain significant concentrations of monovalent copper ions in the cytoplasm. This study demonstrates the effect of monovalent and divalent copper ions on DNA polymerase, ligaseT4 DNA, the restriction enzymes EcoP15I and EcoR I, acid phosphatase, and α and βamylase enzymes. These systems were chosen because they can be monitored under conditions necessary for maintaining a stable concentration of monovalent copper ions, and since they exhibit a wide range of dependency on ATP. Previous studies indicated that ATP interacts with monovalent and divalent copper ions and stabilizing monovalent copper ions over divalent copper ions. The results showed that monovalent copper ions dramatically inhibit DNA polymerase and acid phosphatase, inhibit ligaseT4 DNA and the restriction enzyme EcoP15I, moderately inhibit α and β amylase, and have no effect on the restriction enzyme EcoR I. From the results presented in this work, it can be concluded that the mechanism is not one of oxidative stress, even though monovalent copper ions generate reactive oxygen species (ROS). Molecular oxygen in the medium, which is supposed to increase the oxidative stress, impairs the inhibitory effect of monovalent and divalent copper ions, and the kinetics of the inhibition is not suitable for the ROS mechanism.ATP forms a complex with copper ions (di and monovalent ions, where the latter is more stable) in which the metal ion is bound both to the nitrogen base and to the oxygen charged on the phosphate groups, forming an unusually distorted complex. The results of this study indicate that these complexes have the ability to inhibit enzymatic systems that are dependent on ATP.This finding can provide an explanation for the strong antimicrobial activity of monovalent copper ions, suggesting that rapid and lethal metabolic damage is the main mechanism of monovalent copper ions’ antimicrobial effect.

Fermentation of Dairy-Relevant Sugars by Saccharomyces, Kluyveromyces, and Brettanomyces: An Exploratory Study with Implications for the Utilization of Acid Whey, Part I

Acid whey from Greek-style yogurt (YAW) is an underutilized byproduct and a challenge for the dairy industry. One alternative is the fermentation of YAW by yeasts such as Saccharomyces, Brettanomyces, and Kluyveromyces spp., to produce new styles of fermented beverages. Previous research in our group suggested that the sugar profiles of the dairy coproducts impacted the fermentation profiles produced by B. claussenii. The present work aims to describe the fermentation of dairy sugars by S. cerevisiae, K. marxianus, and B. claussenii, under conditions comparable to those of YAW. For this purpose, four preparations of yeast nitrogen base, each containing 40 g/L of either lactose (LAC), glucose (GLU), galactose (GAL), or a 1:1 mixture of glucose and galactose (GLU:GAL), all at pH 4.20, were used as fermentation media. The fermentation was performed independently by each organism at 25 °C under anoxic conditions, while density, pH, cell count, ethanol, and organic acids were monitored. Non-linear modeling was used to characterize density curves, and Analysis of Variance and Tukey’s Honest Significant Difference tests were used to compare fermentation products. K. marxianus and S. cerevisiae displayed rapid sugar consumption with consistent ethanol yields in all media, as opposed to B. claussenii, which showed more variable results. The latter organism exhibited what appears to be a selective glucose fermentation in GLU:GAL, which will be explored in the future. These results provide a deeper understanding of dairy sugar utilization by relevant yeasts, allowing for future work to optimize fermentations to improve value-added beverage and ingredient production from YAW.

Insulin Regulation of Escherichia coli Abiotic Biofilm Formation: Effect of Nutrients and Growth Conditions

Escherichia coli plays an important role in biofilm formation across a wide array of disease and ecological settings. Insulin can function as an adjuvant in the regulation of biofilm levels. The modulation of insulin-regulated biofilm formation by environmental conditions has not been previously described. In the present study, the effects that various environmental growth conditions and nutrients have on insulin-modulated levels of biofilm production were measured. Micropipette tips were incubated with E. coli ATCC® 25922™ in a Mueller Hinton broth (MH), or a yeast nitrogen base with 1% peptone (YNBP), which was supplemented with glucose, lactose, galactose and/or insulin (Humulin®-R). The incubation conditions included a shaking or static culture, at 23 °C or 37 °C. After incubation, the biofilm production was calculated per CFU. At 23 °C, the presence of insulin increased biofilm formation. The amount of biofilm formation was highest in glucose > galactose >> lactose, while the biofilm levels decreased in shaking cultures, except for galactose (3-fold increase; 0.1% galactose and 20 μU insulin). At 37 °C, regardless of condition, there was more biofilm formation/CFU under static conditions in YNBP than in MH, except for the MH containing galactose. E. coli biofilm formation is influenced by aeration, temperature, and insulin concentration in combination with the available sugars.

The Electrospray (ESI) and Flowing Atmosphere-Pressure Afterglow (FAPA) Mass Spectrometry Studies of Nitrophenols (Plant Growth Stimulants) Removed Using Strong Base-Functionalized Materials

The functional silica-based materials functionalized with a strong nitrogen base TBD (SiO2-TBD) deposited via a linker or with a basic poly(amidoamine) dendrimer containing multiple terminal amine groups -NH2 (SiO2-EDA) and functional polymers containing a strong phosphazene base (Polymer-Phosphazene) or another basic poly(amidoamine) dendrimer (PMVEAMA-PAMAM) were tested as sorbents dedicated to a mixture of nitrophenols (p-nitrophenol and 2-methoxy-5-nitrophenol), which are analogs of nitrophenols used in plant growth biostimulants. The adsorptive potential of the studied materials reached 0.102, 0.089, 0.140, and 0.074 g of the nitrophenols g−1, for SiO2-TBD, SiO2-EDA, polymer-phosphazene, and PMVEAMA-PAMAM, respectively. The sorptive efficiency of the analytes, i.e., their adsorption on the functional materials, the desorption from the obtained [(sorbent)H+ − nitrophenolates–] complexes, and interactions with the used soil, were monitored using mass spectrometry (MS) technique with electrospray (ESI) and flowing atmosphere-pressure afterglow (FAPA) ionizations, for the analysis of the aqueous solutions and the solids, respectively. The results showed that the adsorption/desorption progress is determined by the structures of the terminal basic domains anchored to the materials, which are connected with the strength of the proton exchange between the sorbents and nitrophenols. Moreover, the conducted comprehensive MS analyses, performed for both solid and aqueous samples, gave a broad insight into the interactions of the biostimulants and the presented functional materials.

Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

DNA Finger-Printing: Current Scenario and Future

Linearly arranged chemical structure in chromosome is known as DNA. It is a double helix made up of two strands of genetic material spiraled around each other. Each strand has a sequence of bases. There are four types of basis namely adenine, guanine, cytosine and thiamine which are very unique to each individual just like their actual fingerprint. The nitrogen base adenine always binds with thymine and cytosine also always binds with guanine. Thus the DNA profiling unique to each individual is collectively known as DNA fingerprinting. DNA determines individuality or uniqueness of the each human being except in uniovular twins. The chances of complete similarity are one in 30 billion to 300 billion i.e. half the population of world. The technique of DNA fingerprinting was first developed by Dr. Alec Jeffery’s from Britain in 1984. He discovered a minisatellite region close to the human myoglobin gene. He isolated this sequence and used it as a probe to investigate human DNA. He found that the minisatellite probe result was a complex band pattern for each individual. In India, initially it was done at CCMB, Hyderabad by Dr. Lalji Singh. Now there are various centers where DNA fingerprinting is carried out. In Maharashtra it is carried out at Sate Forensic Science Laboratory, Vidya Nagar, Kalina, Mumbai – 400 098 (Phone 022–26670755). Using this technique FBI formally concluded the participation of Mr. Bill Clinton in Monica Lewyninskey case. In India more than 79 cases have been solved by using this technique including important case of Dhanu and Shivarasan alleged assailant of Late Priminister Shr. Rajiv Gandhi, Tandori case, Madhumati murder case etc.

Counter-Acting Candida albicans-Staphylococcus aureus Mixed Biofilm on Titanium Implants Using Microbial Biosurfactants

This study aimed to grow a fungal-bacterial mixed biofilm on medical-grade titanium and assess the ability of the biosurfactant R89 (R89BS) coating to inhibit biofilm formation. Coated titanium discs (TDs) were obtained by physical absorption of R89BS. Candida albicans-Staphylococcus aureus biofilm on TDs was grown in Yeast Nitrogen Base, supplemented with dextrose and fetal bovine serum, renewing growth medium every 24 h and incubating at 37 °C under agitation. The anti-biofilm activity was evaluated by quantifying total biomass, microbial metabolic activity and microbial viability at 24, 48, and 72 h on coated and uncoated TDs. Scanning electron microscopy was used to evaluate biofilm architecture. R89BS cytotoxicity on human primary osteoblasts was assayed on solutions at concentrations from 0 to 200 μg/mL and using eluates from coated TDs. Mixed biofilm was significantly inhibited by R89BS coating, with similar effects on biofilm biomass, cell metabolic activity and cell viability. A biofilm inhibition >90% was observed at 24 h. A lower but significant inhibition was still present at 48 h of incubation. Viability tests on primary osteoblasts showed no cytotoxicity of coated TDs. R89BS coating was effective in reducing C. albicans-S. aureus mixed biofilm on titanium surfaces and is a promising strategy to prevent dental implants microbial colonization.

D-Fructose Assimilation and Fermentation by Yeasts Belonging to Saccharomycetes: Rediscovery of Universal Phenotypes and Elucidation of Fructophilic Behaviors in Ambrosiozyma platypodis and Cyberlindnera americana

The purpose of this study was to investigate the ability of ascomycetous yeasts to assimilate/ferment d-fructose. This ability of the vast majority of yeasts has long been neglected since the standardization of the methodology around 1950, wherein fructose was excluded from the standard set of physiological properties for characterizing yeast species, despite the ubiquitous presence of fructose in the natural environment. In this study, we examined 388 strains of yeast, mainly belonging to the Saccharomycetes (Saccharomycotina, Ascomycota), to determine whether they can assimilate/ferment d-fructose. Conventional methods, using liquid medium containing yeast nitrogen base +0.5% (w/v) of d-fructose solution for assimilation and yeast extract-peptone +2% (w/v) fructose solution with an inverted Durham tube for fermentation, were used. All strains examined (n = 388, 100%) assimilated d-fructose, whereas 302 (77.8%) of them fermented d-fructose. In addition, almost all strains capable of fermenting d-glucose could also ferment d-fructose. These results strongly suggest that the ability to assimilate/ferment d-fructose is a universal phenotype among yeasts in the Saccharomycetes. Furthermore, the fructophilic behavior of Ambrosiozyma platypodis JCM 1843 and Cyberlindnera americana JCM 3592 was characterized by sugar consumption profiles during fermentation.

Neonatal sepsis due to non-albicans Candida species and their susceptibility to antifungal agents: first report from Bangladesh

Background and objectives: Frequency of neonatal sepsis in Neonatal Intensive Care Units (NICU) has been increasing worldwide over the last decades. The emergence of non-albicans Candida (NAC) species and their resistance to common antifungal agents become an important preventive and therapeutic issue. The present study was undertaken to find out the role of NAC species in neonatal sepsis/candidemia in the NICUs of hospitals of Dhaka city. The susceptibility pattern of NAC species to antifungal agents was also determined. Materials and methods: Suspected cases of neonatal sepsis admitted in NICU of four tertiary care hospitals of Dhaka city, from March to December 2018 were enrolled. In this cross sectional study, blood samples were collected from neonates with suspected sepsis for culture. Identification of Candida species was done by carbohydrate (CHO) assimilation tests using swab auxanographic technique, CHO impregnated yeast nitrogen base plate method (YNB), microtiter plate based miniaturized method and by HiCromeTM Candida Differential Media. Susceptibility of the isolated Candida species to antifungal agents was determined by disk diffusion (DD) and by minimum inhibitory concentration (MIC) methods. MIC was determined by broth microdilution method using RPMI 1640 and trypticase soy broth (TSB). Results: In the present study, NAC species were isolated from 39.7% neonates. C. tropicalis was the predominant species (81.0%) followed by C. parapsilosis (12.1%), C. auris (5.2%) and C. dubliniensis (1.7%). Isolated NAC species were 98.3% sensitive to voriconazole. Sensitivity to fluconazole, ketoconazole, itraconazole, and clotrimazole was 3.5%, 15.5%, 86.2% and 56.9% respectively by DD method. All the isolates (100%) were sensitive to miconazole and nystatin. All the C. tropicalis, C. auris and C. dubliniensis were sensitive to amphotericin B and anidulafungin. One and four C. parapsilosis were found resistant to amphotericin B and anidulafungin respectively. The MIC results obtained by using RPMI 1640 and TSB as growth medium were concordant suggesting that TSB media was a good alternative to expensive RPMI 1640. Conclusion: The advent of NAC species merits attention as they are highly resistant to most of the azoles. Therefore, speciation of Candida in neonatal candidemia is essential to institute appropriate antifungal therapy. Ibrahim Med. Coll. J. 2020; 14(2): 19-26