Pancreatic adenocarcinoma (PA) is an aggressive cancer. It develops in a way that causes almost no detectable symptoms, which leads to a rapid progression and a short survival rate. Researchers have discovered a link between pancreatic cancer (and other cancer types) and obesity. High levels of leptin, an appetite hormone secreted by adipocytes, have been found in obese people. Studies have shown that the absence of leptin in the body or severe leptin resistance can lead to uncontrolled eating and weight gain, hence, its connection to obesity. Consequently, our lab is analyzing the relationship between obesity and leptin and what effects they have on pancreatic cancer progression. We hypothesize that in PA cells, leptin induces proliferation, tumorigenesis, and increased levels of Notch and related molecules. These effects are reversed by our leptin antagonist linked to iron nanoparticles, IONP-LPrA2 (iron oxidized nanoparticles leptin peptide receptor antagonist). We’re mainly focu...

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre...

Poly-lactic acid (PLA) is increasingly used as a biodegradable alternative to traditional petroleum-based plastics. In this study, we identify a novel agricultural soil isolate of Bacillus pumilus (B12) that is capable of degrading high molecular weight PLA films. This degradation can be detected on a short timescale, with significant degradation detected within 48-h by the release of L-lactate monomers, allowing for a rapid identification ideal for experimental variation. The validity of using L-lactate as a proxy for degradation of PLA films is corroborated by loss of rigidity and appearance of fractures in PLA films, as measured by atomic force microscopy and scanning electron microscopy (SEM), respectively. Furthermore, we have observed a dose-dependent decrease in PLA degradation in response to an amino acid/nucleotide supplement mix that is driven mainly by the nucleotide base adenine. In addition, amendments of the media with specific carbon sources increase the rate of PLA degradation, while phosphate and potassium additions decrease the rate of PLA degradation by B. pumilus B12. These results suggest B. pumilus B12 is adapting its enzymatic expression based on environmental conditions and that these conditions can be used to study the regulation of this process. Together, this work lays a foundation for studying the bacterial degradation of biodegradable plastics.

Due to the increased number of immunocompromised patients, infections with the pathogen Candida albicans have significantly increased in recent years. C. albicans transition from yeast to germ tubes is one of the essential factors for virulence. In this study we noted that Lee's medium, commonly used to induce filamentation, contained 500-fold more biotin than needed for growth and 40-fold more biotin than is typically added to growth media. Thus, we investigated the effects of excess biotin on growth rate and filamentation by C. albicans in different media. At 37 • C, excess biotin (4 µM) enhanced germ tube formation (GTF) ca. 10-fold in both Lee's medium and a defined glucose-proline medium, and ca. 4-fold in 1% serum. Two biotin precursors, desthiobiotin and 7-keto-8-aminopelargonic acid (KAPA), also stimulated GTF. During these studies we also noted an inverse correlation between the number of times the inoculum had been washed and the concentration of serum needed to stimulate GTF. C. albicans cells that had been washed eight times achieved 80% GTF with only 0.1% sheep serum. The mechanism by which 1-4 µM biotin enhances GTF is still unknown except to note that equivalent levels of biotin are needed to create an internal supply of stored biotin and biotinylated histones. Biotin did not restore filamentation for any of the four known filamentation defective mutants tested. C. albicans is auxotrophic for biotin and this biotin auxotrophy was fulfilled by biotin, desthiobiotin, or KAPA. However, biotin auxotrophy is not temperature dependent or influenced by the presence of 5% CO 2. Biotin starvation upregulated the biotin biosynthetic genes BIO2, BIO3, and BIO4 by 11-, 1500-, and 150-fold, respectively, and BIO2p is predicted to be mitochondrion-localized. Based on our findings, we suggest that biotin has two roles in the physiology of C. albicans, one as an enzymatic cofactor and another as a morphological regulator. Finally, we found no evidence supporting prior claims that C. albicans only forms hyphae at very low biotin (0.1 nM) growth conditions.

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origen of C106 reactivity is obscure, due in part to the absence of an experimentally determined pK a value for this residue. We have used atomic resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed pK a of 5.4±0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid sidechain (E18). X-ray crystal structures and cysteine pK a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid sidechain of E18 is required to maximally stabilize the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the pK a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48 and R28) surrounding C106 affect its pK a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen bonded aspartic/glutamic acid-cysteine interactions, which we propose are particularly common in the DJ-1 superfamily. Keywords cysteine; pK a ; atomic resolution X-ray crystallography; bond length analysis; Parkinsonism Cysteine thiolates are potent nucleophiles that are used by many proteins for catalysis, metal binding, or to facilitate post-translational modification. However, the solution pK a value of cysteine (8.3) is not within the optimal physiological pH range of most proteins. To render cysteine residues reactive, the thiol pK a must be depressed by stabilization of the conjugate thiolate anion by the protein environment. Multiple types of stabilizing interactions can decrease cysteine pK a values, including electrostatic complementarity with nearby cations (1,2), the α-helix macropole (3), and hydrogen bonding to the thiol (4).

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the se...

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical, and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and-derived bacterial isolates to determine species selectivity. Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE) in the cell membrane. Further the surface and interface morphology were studied by high...

Candida albicans is among the most common human fungal pathogens. The ability to undergo the morphological transition from yeast to hyphal growth is critical for its pathogenesis. Farnesol, a precursor in the isoprenoid/sterol pathway, is a quorum-sensing molecule produced by C. albicans that inhibits hyphal growth in this polymorphic fungus. Interestingly, C. albicans can tolerate farnesol concentrations that are toxic to other fungi. We hypothesized that changes in phospholipid composition are one of the factors contributing to farnesol tolerance in C. albicans. In this study, we found that loss of enzymes that synthesize the phospholipids phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) compromise the tolerance of C. albicans to farnesol. Compared with wild type, the phospholipid mutant cho1∆/∆ (loss of PS and decreased PE synthesis) shows greater inhibition of growth, loss of ATP production, increased consumption of oxygen, and increased formation of reactive oxygen ...

mutants for phosphatidylserine (PS) synthase (ΔΔ) and PS decarboxylase (ΔΔ ΔΔ) are compromised for virulence in mouse models of systemic infection and oropharyngeal Candidiasis (OPC). Both of these enzymes are necessary to synthesize phosphatidylethanolamine (PE) by the pathway, but these mutants are still capable of growth in culture media as they can import ethanolamine from media to synthesize PE through the Kennedy pathway. Given that the host has ethanolamine in its serum, the exact mechanism by which virulence is lost in these mutants is not clear. There are two competing hypotheses to explain their loss of virulence. 1) PE from the Kennedy pathway cannot substitute for synthesized PE. 2) The mutants cannot acquire sufficient ethanolamine from the host to support adequate PE synthesis. These hypotheses can be simultaneously tested if ethanolamine availability is increased for while it is inside the host. We accomplish this by transcomplementation of with the serine decarboxyla...

The emergence of rapidly expanding infectious diseases such as coronavirus (COVID-19) demands effective biosensors that can promptly detect and recognize the pathogens. Field-effect transistors based on semiconducting two-dimensional (2D) materials (2D-FETs) have been identified as potential candidates for rapid and label-free sensing applications. This is because any perturbation of such atomically thin 2D channels can significantly impact their electronic transport properties. Here, we report the use of FET based on semiconducting transition metal dichalcogenide (TMDC) WSe 2 as a promising biosensor for the rapid and sensitive detection of SARS-CoV-2 in vitro. The sensor is created by functionalizing the WSe 2 monolayers with a monoclonal antibody against the SARS-CoV-2 spike protein and exhibits a detection limit of down to 25 fg/μL in 0.01X phosphate-buffered saline (PBS). Comprehensive theoretical and experimental studies, including density functional theory, atomic force microscopy, Raman and photoluminescence spectroscopies, and electronic transport properties, were performed to characterize and explain the device performance. The results demonstrate that TMDC-based 2D-FETs can potentially serve as sensitive and selective biosensors for the rapid detection of infectious diseases.

Candida albicans is an opportunistic fungal pathogen in humans. It is a polymorphic fungus: it can live as yeasts, hyphae, or pseudohyphae. Biotin is required for cell growth and fatty acid metabolism because it is used as a cofactor for carboxylases such as acetyl-CoA carboxylase, and pyruvate carboxylase. In addition, we have discovered that biotin is used to modify histones in C. albicans. Biotinylation was detected by Western blots using a monoclonal antibiotin HRP-conjugated antibody as well as with qTOF and LC/MS/MS mass spectrometry. As a precaution, the antibiotin antibody was dialyzed against neutravidin prior to use. During this study, we observed that three histones, H2A, H2B, and H4, were biotinylated at many lysine residues in an apparently nonsite-specific manner. Roughly, equivalent levels of acetylation, methylation, and phosphorylation were found in histones from biotin-replete and biotin-starved cells, but histone biotinylation was only observed for cells grown in ...

Farnesol interacts with Candida albicans as both a quorum-sensing molecule and toxic agent, but confusion abounds regarding which conditions promote these distinct responses. Farnesol sensitivity was measured when inoculum cell history and size, temperature, and growth media were altered. Parameters for farnesol tolerance/sensitivity were defined, validating previous studies and identifying new variables, such as energy availability. This study clearly defines what farnesol concentrations are lethal to C. albicans, based on environmental conditions.

Candida albicans is an opportunistic fungal pathogen in humans. It is a polymorphic fungus: it can live as yeasts, hyphae, or pseudohyphae. Biotin is required for cell growth and fatty acid metabolism because it is used as a cofactor for carboxylases such as acetyl-CoA carboxylase, and pyruvate carboxylase. In addition, we have discovered that biotin is used to modify histones in C. albicans. Biotinylation was detected by Western blots using a monoclonal antibiotin HRP-conjugated antibody as well as with qTOF and LC/MS/MS mass spectrometry. As a precaution, the antibiotin antibody was dialyzed against neutravidin prior to use. During this study, we observed that three histones, H2A, H2B, and H4, were biotinylated at many lysine residues in an apparently nonsite-specific manner. Roughly, equivalent levels of acetylation, methylation, and phosphorylation were found in histones from biotin-replete and biotin-starved cells, but histone biotinylation was only observed for cells grown in excess biotin. The function of histone biotinylation in C. albicans is still unknown but, because C. albicans is a natural biotin auxotroph, a storage reservoir for biotin is attractive. Techniques used to detect histone biotinylation in C. albicans did not detect any histone biotinylation in Saccharomyces cerevisiae.

Background: Glyoxalases are a varied group of enzymes that detoxify methylglyoxal by converting it to D-lactate. Results: The Candida albicans glyoxalase Glx3 is important for yeast growth, especially in glycerol. Conclusion: Many yeasts contain a novel group of glyoxalases that are not redundant with previously characterized enzymes. Significance: This is the first demonstration of physiologically relevant glutathione-independent glyoxalases in fungi. Methylglyoxal is a cytotoxic reactive carbonyl compound produced by central metabolism. Dedicated glyoxalases convert methylglyoxal to D-lactate using multiple catalytic strategies. In this study, the DJ-1 superfamily member ORF 19.251/GLX3 from Candida albicans is shown to possess glyoxalase activity, making this the first demonstrated glutathione-independent glyoxalase in fungi. The crystal structure of Glx3p indicates that the protein is a monomer containing the catalytic triad Cys 136-His 137-Glu 168. Purified Glx3p has an in vitro methylglyoxalase activity (K m ‫؍‬ 5.5 mM and k cat ‫؍‬ 7.8 s ؊1) that is significantly greater than that of more distantly related members of the DJ-1 superfamily. A close Glx3p homolog from Saccharomyces cerevisiae (YDR533C/Hsp31) also has glyoxalase activity, suggesting that fungal members of the Hsp31 clade of the DJ-1 superfamily are all probable glutathione-independent glyoxalases. A homozygous glx3 null mutant in C. albicans strain SC5314 displays greater sensitivity to millimolar levels of exogenous methylglyoxal, elevated levels of intracellular methylglyoxal, and carbon source-dependent growth defects, especially when grown on glycerol. These phenotypic defects are complemented by restoration of the wild-type GLX3 locus. The growth defect of Glx3-deficient cells in glycerol is also partially complemented by added inorganic phosphate, which is not observed for wild-type or glucose-grown cells. Therefore, C. albicans Glx3 and its fungal homologs are physiologically relevant glutathione-independent glyoxalases that are not redundant with the previously characterized glutathione-dependent GLO1/GLO2 system. In addition to its role in detoxifying glyoxals, Glx3 and its close homologs may have other important roles in stress response.

Farnesol has been best studied for its role in regulating fungal dimorphism. However, farnesol is also a lipid and in this review we analyze data relevant to farnesol's function and synthesis from the perspective of farnesol and bacterial endotoxins acting as membrane active compounds. This analysis implicates the possible roles of: (1) endotoxins in the regulation of farnesol production by C. albicans; (2) farnesol in the interactions between C. albicans and the host during disseminated infections; and (3) ubiquinones in the mechanisms for unusually high resistance to farnesol by some C. albicans cell types. Finally we discuss the implications that the use of farnesol as both a signaling molecule and to antagonize competing microbials species has for the regulation of HMG-CoA reductase, the enzyme that is the usual rate limiting step in sterol/ lipid synthesis.

Farnesol interacts with Candida albicans as both a quorum-sensing molecule and toxic agent, but confusion abounds regarding which conditions promote these distinct responses. Farnesol sensitivity was measured when inoculum cell history and size, temperature, and growth media were altered. Parameters for farnesol tolerance/ sensitivity were defined, validating previous studies and identifying new variables, such as energy availability. This study clearly defines what farnesol concentrations are lethal to C. albicans, based on environmental conditions

Candida albicans is an opportunistic fungal pathogen in humans. It is a polymorphic fungus: it can live as yeasts, hyphae, or pseudohyphae. Biotin is required for cell growth and fatty acid metabolism because it is used as a cofactor for carboxylases such as acetyl-CoA carboxylase, and pyruvate carboxylase. In addition, we have discovered that biotin is used to modify histones in C. albicans. Biotinylation was detected by Western blots using a monoclonal antibiotin HRP-conjugated antibody as well as with qTOF and LC/MS/MS mass spectrometry. As a precaution, the antibiotin antibody was dialyzed against neutravidin prior to use. During this study, we observed that three histones, H2A, H2B, and H4, were biotinylated at many lysine residues in an apparently nonsite-specific manner. Roughly, equivalent levels of acetylation, methylation, and phosphorylation were found in histones from biotin-replete and biotin-starved cells, but histone biotinylation was only observed for cells grown in excess biotin. The function of histone biotinylation in C. albicans is still unknown but, because C. albicans is a natural biotin auxotroph, a storage reservoir for biotin is attractive. Techniques used to detect histone biotinylation in C. albicans did not detect any histone biotinylation in Saccharomyces cerevisiae.

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origen of C106 reactivity is obscure, due in part to the absence of an experimentally determined pK a value for this residue. We have used atomic resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed pK a of 5.4±0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid sidechain (E18). X-ray crystal structures and cysteine pK a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid sidechain of E18 is required to maximally stabilize the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the pK a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48 and R28) surrounding C106 affect its pK a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen bonded aspartic/glutamic acid-cysteine interactions, which we propose are particularly common in the DJ-1 superfamily. Keywords cysteine; pK a ; atomic resolution X-ray crystallography; bond length analysis; Parkinsonism Cysteine thiolates are potent nucleophiles that are used by many proteins for catalysis, metal binding, or to facilitate post-translational modification. However, the solution pK a value of cysteine (8.3) is not within the optimal physiological pH range of most proteins. To render cysteine residues reactive, the thiol pK a must be depressed by stabilization of the conjugate thiolate anion by the protein environment. Multiple types of stabilizing interactions can decrease cysteine pK a values, including electrostatic complementarity with nearby cations (1,2), the α-helix macropole (3), and hydrogen bonding to the thiol (4).

In order to determine the prevalence of metabolic syndrome (MS) and its individual components in children and adolescents from three parishes of the Municipality of Maracaibo, Zulia State; 501 subjects, from 10 to 16 years old and both sexes were selected. The children and adolescents were evaluated clinical, anthropometric and biochemically. For the diagnosis of MS three definitions were used which have been modified for children and adolescents: By using the definition of the International Diabetes Federation (IDF) the prevalence was 2.99%, 6.18% with the criteria Da Silva et al. and 8.58% with the criteria of Cook et al. Most of those diagnosed with MS, were in the overweight and obese group, affecting even subjects with normal body mass index (BMI). BMI and waist circumference (CC) showed significant positive correlation with weight, height, basal insulin (InsBas), LDL-c, VLDL-c, total cholesterol (TC), arm circumference (AC) and body fat percentage (%FM). The basal blood glucos...