Resistance in Candida albicans: exploring the cell wall barrier by proteomics

The incidence of candidiasis has dramatically increased and bloodstream infections due to different species of Candida are becoming a prime cause of morbidity and mortality in different types of immunocompromised patients. Azole and echinocandin drug resistance accounts for the dramatic increase in incidence of nosocomial bloodstream candidiasis found in recent years. Cell wall constitutes the barrier between the yeast and the host and resistant strains change the proteome of this compartment. In the last decade different proteomic platforms have been applied to study cell wall and markers of resistance to drugs have been pointed out. Modulation of these proteins seem to suggest that although resistance is based on a specific mutation able to counteract the toxicity of the antifungal drug, a set of other molecular modifications takes place contemporary or subsequently the establishment of the resistance and seems to support the viability of the resistant yeast. Profiled proteins by proteomics may be valuable in design therapy using classical antifungal along with complementary drugs able to abolish pathways that strengthen the resistance and attenuate virulence of the mutated cell

STAVOVI UČITELJA RAZREDNE NASTAVE O STRUČNIM USAVRŠAVANJIMA

Stručna usavršavanja sastavni su dio profesionalnog razvoja svakog odgojno- obrazovnog djelatnika. Razlikuju se vrste i načini stručnih usavršavanja. Kako bi ona bila korisna učiteljima važan je uvid u učiteljska razmišljanja, stavove i doživljaje o dosadašnjim usavršavanjima koja su pohađali. Iz navedenog razloga provedeno je kvalitativno istraživanje polustrukturiranim intervjuom na prigodnom uzorku od 12 učitelja. Iz dobivenih podataka vidljivo je kako učitelji imaju različite motivatore za stručnim usavršavanjima. Najzastupljeniji oblik stručnih usavršavanja navode se formalna stručna usavršavanja. Ispitanici kao negativne aspekte takvog načina usavršavanja navode neaktualnu tematiku te slabu metodičku pripremu predavača. Iz odgovora ispitanika vidljiv je prostor za napredak sustavnosti provedbe i organizacije stručnih usavršavanja

A chromophore in glutamate decar☐ylase has been wrongly identified as PQQ

AbstractPyrroloquinoline quinone (PQQ) has been claimed to be a component of glutamate decar☐ylase fromEscherichia coli on the basis of a frequently used procedure in which the protein is extracted with hexanol. We demonstrate that if pyridoxal phosphate (PLP) is not added during the preparation, the apoenzyme prepared from glutamate decar☐ylase contains no chromophore absorbing above 280 nm. Full enzyme activity and the original holoenzyme spectrum are restored by the addition of PLP alone, A 340 nm-absorbing band, similar to that which prompted analysis for PQQ, is produced by exposure of the enzyme to solutions of PLP

Membrane protein remodeling in microglia exposed to amyloid peptides

Infection, neurodegeneration, and other conditions associated with loss of brain homeostasis, induce changes in microglial morphology, gene expression and function, generally referred to as “activation”. Alzheimer’s disease (AD) is the most common dementia and is characterized by neuroinfammatory changes, including alterations in the morphology and distribution of microglia and astrocytes, and deposition of complement and other infammatory mediators. Our previous observations show that microglial cells challenged in vitro with amyloid peptides clustered and rounded up, dramatically changing their morphology. Besides, in these cells we observed the early acetylation and then the phosphorylation of STAT3 which is required for the expression of the epsilon isoform of 14-3-3, a marker of Abeta-activated microglia (1, 2). We applied afnity partitioning approach combined with high throughput mass spectrometric analysis in order to identify variation of proteins on plasma membrane of BV2 immortalized microglia upon treatment with amyloid peptides. By this method several proteins up- or down-regulated by amyloid treatment were identifed in microglial plasma membrane. Among them annexins (5 and 7), IFITM3 and MARK3. These data have been confrmed in primary microglial cultures. In microglia, plasma membrane plays a relevant role in the cross-talking with the external neuronal environment and in the resulting trophic or infammatory response of these sentinel cells. As such, knowledge of the microglia responsiveness to beta amyloids in term of changes in its plasma membrane proteome is imperative for unveiling the molecular landscape in which AD occurs

Hyperexpression of CDRs and HWP1 genes negatively impacts on Candida albicans virulence

C. albicans is a commensal organism present in the human microbiome of more than 60% of the healthy population. Transition from commensalism to invasive candidiasis may occur after a local or a general failure of host's immune system. This transition to a more virulent phenotype may reside either on the capacity to form hyphae or on an acquired resistance to antifungal drugs. Indeed, overexpression of genes coding drug efflux pumps or adhesins, cell wall proteins facilitating the contact between the fungus and the host, usually marks the virulence profile of invasive Candida spp. In this paper, we compare virulence of two clinical isolates of C. albicans with that of laboratory-induced resistant strains by challenging G. mellonella larvae with these pathogens along with monitoring transcriptional profiles of drug efflux pumps genes CDR1, CDR2, MDR1 and the adhesin genes ALS1 and HWP1. Although both clinical isolates were found resistant to both fluconazole and micafungin they were found less virulent than laboratory-induced resistant strains. An unexpected behavior emerged for the former clinical isolate in which three genes, CDR1, CDR2 and HWP1, usually correlated with virulence, although hyperexpressed, conferred a less aggressive phenotype. On the contrary, in the other isolate, we observed a decreased expression of CDR1, CDR2 and HWP1as well as of MDR1 and ALS1 that may be consistent with the less aggressive performance observed in this strain. These altered gene expressions might directly influence Candida virulence or they might be an epiphenomenon of a vaster rearrangement occurred in these strains during the challenge with the host's environment. An in-deepth comprehension of this scenario could be crucial for developing interventions able to counteract C. albicans invasiveness and lethality.C. albicans is a commensal organism present in the human microbiome of more than 60% of the healthy population. Transition from commensalism to invasive candidiasis may occur after a local or a general failure of host's immune system. This transition to a more virulent phenotype may reside either on the capacity to form hyphae or on an acquired resistance to antifungal drugs. Indeed, overexpression of genes coding drug efflux pumps or adhesins, cell wall proteins facilitating the contact between the fungus and the host, usually marks the virulence profile of invasive Candida spp. In this paper, we compare virulence of two clinical isolates of C. albicans with that of laboratory-induced resistant strains by challenging G. mellonella larvae with these pathogens along with monitoring transcriptional profiles of drug efflux pumps genes CDR1, CDR2, MDR1 and the adhesin genes ALS1 and HWP1. Although both clinical isolates were found resistant to both fluconazole and micafungin they were found less virulent than laboratory-induced resistant strains. An unexpected behavior emerged for the former clinical isolate in which three genes, CDR1, CDR2 and HWP1, usually correlated with virulence, although hyperexpressed, conferred a less aggressive phenotype. On the contrary, in the other isolate, we observed a decreased expression of CDR1, CDR2 and HWP1as well as of MDR1 and ALS1 that may be consistent with the less aggressive performance observed in this strain. These altered gene expressions might directly influence Candida virulence or they might be an epiphenomenon of a vaster rearrangement occurred in these strains during the challenge with the host's environment. An in-deepth comprehension of this scenario could be crucial for developing interventions able to counteract C. albicans invasiveness and lethality

Primary structure and tetrahydropteroylglutamate binding site of rabbit liver cytosolic 5,10-methenyltetrahydrofolate synthetase.

The primary sequence of 5,10-methenyltetrahydrofolate synthetase from rabbit liver was determined by amino acid sequencing of the purified enzyme. The enzyme contains 201 amino acid residues with a predicted mass of 22,779 Da. The enzyme is located in the cytosolic fraction of liver homogenates. Carbodiimide-activated 5-formyltetrahydropteroylmonoglutamate and the pentaglutamate form of the substrate both irreversibly inactivate the enzyme by forming a covalent bond to Lys-18. Non-activated 5-formyltetrahydropteroylpentaglutamate protected against this inactivation. Substrate specificity studies showed that increasing the number of glutamate residues from zero to five on 5-formyltetrahydropteroate results in a 2 order of magnitude increase in the affinity of the substrate for the enzyme but only a 3-fold increase in the value of Vmax

E-vrednovanje u nastavi na daljinu tijekom pandemije COVID-19 iz perspektive učitelja

Pandemija virusa COVID-19 u određenom je vremenu promijenila način funkcioniranja odgojno-obrazovnih sustava. Nastava je provođena na daljinu što je učiteljima donijelo brojne izazove, a jedan od njih bilo je e-vrednovanje. Ovaj rad daje uvid u učiteljsko viđenje problematike provedbe e-vrednovanja tijekom nastave na daljinu. Za potrebe prikupljanja podataka u ovom je istraživanju korišten polustrukturirani intervju. Sudjelovalo je 16 učitelja obaju spolova. Prema rezultatima, razvidno je da e-vrednovanje učiteljima predstavlja izazov. Posebno se istaknula problematika učeničke samostalnosti i razine usvojenosti nastavnih sadržaja što dovodi u pitanje svrhovitost i relevantnost e-vrednovanja

E-vrednovanje u nastavi na daljinu tijekom pandemije COVID-19 iz perspektive učitelja

Pandemija virusa COVID-19 u određenom je vremenu promijenila način funkcioniranja odgojno-obrazovnih sustava. Nastava je provođena na daljinu što je učiteljima donijelo brojne izazove, a jedan od njih bilo je e-vrednovanje. Ovaj rad daje uvid u učiteljsko viđenje problematike provedbe e-vrednovanja tijekom nastave na daljinu. Za potrebe prikupljanja podataka u ovom je istraživanju korišten polustrukturirani intervju. Sudjelovalo je 16 učitelja obaju spolova. Prema rezultatima, razvidno je da e-vrednovanje učiteljima predstavlja izazov. Posebno se istaknula problematika učeničke samostalnosti i razine usvojenosti nastavnih sadržaja što dovodi u pitanje svrhovitost i relevantnost e-vrednovanja

Mechanism-based Inactivation of Dopa Decarboxylase by Serotonin

Abstract Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5′-phosphate active site per subunit. In addition to catalyzing the decarboxylation of L-aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde and ammonia. These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high performance liquid chromatographic and mass spectroscopic analysis. The Kcat and Km values of this reaction were determined to be 0.48 min−1 and 0.47 mM, respectively. The NaBH4-reduced enzyme does not catalyze this reaction. Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with Ki and Kinact values of 0.40 mM and 0.023 min−1, respectively. Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine. Inactivation with [2-14C]5-HT results in the incorporation of 1 mol of label/enzyme subunit. Taken together, these findings indicate that 5-HT is both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21. The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized. A speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented

The primary structure of mitochondrial aspartate aminotrasferase from human heart

The complete amino acid sequence of the mitochondrial asparate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained