Mucormycosis is a lethal and difficult-to-treat fungal infection caused by fungi of the order Mucorales.
Mucor lusitanicus,
a member of Mucorales, is commonly used as a model to understand disease ...pathogenesis. However, transcriptional control of hyphal growth and virulence in Mucorales is poorly understood. This study aimed to investigate the role of Tec proteins, which belong to the TEA/ATTS transcription factor family, in the hyphal development and virulence of
M. lusitanicus
. Unlike in the genome of Ascomycetes and Basidiomycetes, which have a single Tec homologue, in the genome of Mucorales, two Tec homologues, Tec1 and Tec2, were found, except in that of
Phycomyces blakesleeanus
, with only one Tec homologue.
tec1
and
tec2
overexpression in
M. lusitanicus
increased mycelial growth, mitochondrial content and activity, expression of the rhizoferrin synthetase-encoding gene
rfs
, and virulence in nematodes and wax moth larvae but decreased cAMP levels and protein kinase A (PKA) activity. Furthermore,
tec1
- and
tec2
-overexpressing strains required adequate mitochondrial metabolism to promote the virulent phenotype. The heterotrimeric G beta subunit 1-encoding gene deletant strain (Δ
gpb1
) increased cAMP-PKA activity, downregulation of both
tec
genes, decreased both virulence and hyphal development, but
tec1
and
tec2
overexpression restored these defects. Overexpression of allele-mutated variants of Tec1(S332A) and Tec2(S168A) in the putative phosphorylation sites for PKA increased both virulence and hyphal growth of Δ
gpb1
. These findings suggest that Tec homologues promote mycelial development and virulence by enhancing mitochondrial metabolism and rhizoferrin accumulation, providing new information for the rational control of the virulent phenotype of
M. lusitanicus
.
Evidence suggests that histone deacetylases (HDACs) inhibitors could be used as an effective treatment for some psychiatric and neurological conditions such as depression, anxiety and age-related ...cognitive decline. However, non-specific HDAC inhibiting compounds have a clear disadvantage regarding their efficacy and safety, thus the need to develop more selective ones. The present study evaluated the toxicity, the capacity to inhibit HDAC activity and antidepressant-like activity of three recently described class I HDAC inhibitors IN01, IN04 and IN14, using
toxicity test,
fluorometric HDAC activity assay and forced-swimming test, respectively. Our data show that IN14 possesses a better profile than the other two. Therefore, the pro-cognitive and antidepressant effects of IN14 were evaluated. In the forced-swimming test model of depression, intraperitoneal administration of IN14 (100 mg/Kg/day) for five days decreased immobility, a putative marker of behavioral despair, significantly more than tricyclic antidepressant desipramine, while also increasing climbing behavior, a putative marker of motivational behavior. On the other hand, IN14 left the retention latency in the elevated T-maze unaltered. These results suggest that novel HDAC class I inhibitor IN14 may represent a promising new antidepressant with low toxicity and encourages further studies on this compound.
Human societies demand sustainable alternatives for goods and services. Plants are sustainable sources of important metabolites with beneficial impacts on human health. There are many reported ...methodologies and commercial suppliers for extract preparations from Mucuna sp. They usually claim the plant is enriched with L-dopa, its distinctive metabolite. However, many present poor characterizations of the extract’s components. Here, we present polar metabolites characterization of a Mucuna seed extract, emphasizing L-dopa identification and quantification. To obtain the extracts, we follow a green and sustainable extraction protocol. The lyophilized extract is subjected to liquid chromatography and mass spectrometry to identify its primary metabolites. Additionally, we follow thin-layer chromatography to identify carbohydrates in the sample. The resultant extract has 56% L-dopa. Other main components in the extract are arginine, stizolamine, and the fructooligosaccharides sucrose and nystose. The characterized Mucuna extract can be easily standardized using powder preparation and used in several biomedical applications.
This study analyzed the role of blood serum in enhancing the mitochondrial metabolism and virulence of Mucorales through rhizoferrin secretion. We observed that the spores of clinically relevant ...Mucorales produced in the presence of serum exhibited higher virulence in a heterologous infection model of
. Cell-free supernatants of the culture broth obtained from spores produced in serum showed increased toxicity against
, which was linked with the enhanced secretion of rhizoferrin. Spores from Mucoralean species produced or germinated in serum showed increased respiration rates and reactive oxygen species levels. The addition of non-lethal concentrations of potassium cyanide and N-acetylcysteine during the aerobic or anaerobic growth of Mucorales decreased the toxicity of the cell-free supernatants of the culture broth, suggesting that mitochondrial metabolism is important for serum-induced virulence. In support of this hypothesis, a mutant strain of
that lacks fermentation and solely relies on oxidative metabolism exhibited virulence levels comparable to those of the wild-type strain under serum-induced conditions. Contrary to the lower virulence observed, even in the serum, the ADP-ribosylation factor-like 2 deletion strain exhibited decreased mitochondrial activity. Moreover, spores produced in the serum of
and
that grew in the presence of a mitophagy inducer showed low virulence. These results suggest that serum-induced mitochondrial activity increases rhizoferrin levels, making Mucorales more virulent.
The non-appropriate conditions faced by nutritionally stressed bacteria propitiate error-prone repair events underlying stationary-phase- or stress-associated mutagenesis (SPM). The genetic and ...molecular mechanisms involved in SPM have been deeply studied but the biochemical aspects of this process have so far been less explored. Previous evidence showed that under conditions of nutritional stress, non-dividing cells of strain
B. subtilis
YB955 overexpressing ribonucleotide reductase (RNR) exhibited a strong propensity to generate true reversions in the
hisC952
(amber),
metB5
(ochre) and
leuC425
(missense) mutant alleles. To further advance our knowledge on the metabolic conditions underlying this hypermutagenic phenotype, a high-throughput LC–MS/MS proteomic analysis was performed in non-dividing cells of an amino acid-starved strain, deficient for NrdR, the RNR repressor. Compared with the parental strain, the level of 57 proteins was found to increase and of 80 decreases in the NrdR-deficient strain. The proteomic analysis revealed an altered content in proteins associated with the stringent response, nucleotide metabolism, DNA repair, and cell signaling in amino acid-starved cells of the ∆
nrdR
strain. Overall, our results revealed that amino acid-starved cells of strain
B. subtilis
∆
nrdR
that escape from growth-limiting conditions exhibit a complex proteomic pattern reminiscent of a disturbed metabolism. Future experiments aimed to understand the consequences of disrupting the cell signaling pathways unveiled in this study, will advance our knowledge on the genetic adaptations deployed by bacteria to escape from growth-limiting environments.
Herein we report the mild synthesis of 15 phosphonocinnamic esters by means of a solvent-free mechanically activated Horner-Wadsworth-Emmons (HWE) olefination, in regular to excellent yields ...(13-99%), from tetraethyl methylenediphosphonate, an aromatic or aliphatic aldehyde and potassium tert-butoxide, using 5 min of mortar and pestle grinding. Potassium tert-butoxide acts as a base and liquid-assisting grinding agent. All isolated products were fully characterized as (E)-isomers through NMR spectroscopy. Furthermore, among the synthesized compounds diethyl (E)-2-(4-methoxyphenyl)vinylphosphonate 1 was discovered as an interesting cytotoxic agent inhibiting growth of two breast cancer cell lines (MDA-MB-453, IC
50
= 79 µg/mL and MCF-7 IC
50
= 64 µg/mL). To the best of our knowledge, this is the first report of mechanically activated HWE reaction for the synthesis of α, β-unsaturated phosphonates and the first time some of them have been assessed as cytotoxic agents on breast cancer cell lines. In conclusion, the fast and operationally simple synthesis of (E)-phosphonocinnamic esters afforded new promising cytotoxic agents in regular to excellent yields in very short reaction times.
The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, ...we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-β-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H
O
and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM.
Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions.
The analysis of the global DNA methylation, calculated as the percentage of 5-methylcytosine (5mC) over the total sum of cytosines, is a well stablished biomarker for monitoring large scale ...epigenetic events in organisms. DNA purification, hydrolysis, separation and detection methods are critical steps to determine this biomarker. In the present work is proposed a robust procedure for DNA acid-hydrolysis assisted by microwave that provides identical DNA methylation patterns that enzymatic hydrolysis and better release of 5mC than acid classic method. The quantification was performed using a gas chromatographer coupled to a mass spectrometer with triple quadrupole as mass analyzer (GC-TQ-MS/MS) using multiple reaction monitoring (MRM) mode for the trimethylsilyl-derivates of nucleobases; following the transitions of 254→238, 240→170 and 254→238, 254→184 (m/z) for C and 5mC respectively, was achieved a limit of detection of 0.46 fmol for C and 0.41 fmol for 5mC. The proposed procedure is capable of determine 0.004% of 5mC in 50 ng of DNA in a chromatographic time of 10 minutes, being a good alternative to LC-MS/MS analysis.
Elucidating how a population of non-growing bacteria generates mutations improves our understanding of phenomena like antibiotic resistance, bacterial pathogenesis, genetic diversity and evolution. ...To evaluate mutations that occur in nutritionally stressed non-growing bacteria, we have employed the strain
YB955, which measures the reversions rates to the chromosomal auxotrophies
and
(Sung and Yasbin, 2002). This gain-of-function system has successfully allowed establishing the role played by repair systems and transcriptional factors in stress-associated mutagenesis (SPM) (Barajas- Ornelas
, 2014 ; Gómez- Marroquín
, 2016 ). In a recent study (Castro- Cerritos
, 2017 ), it was found that Ribonucleotide Reductase (RNR) was necessary for SPM; this enzyme is essential in this bacterium. We engineered a conditional mutant of strain
YB955 in which expression of the
operon was modulated by isopropyl-β-D-thiogalactopyranoside (IPTG) (Castro- Cerritos
, 2017 ). The conditions to determine mutation frequencies conferring amino acid prototrophy in three genes (
) under nutritional stress in this conditional mutant are detailed here. This technique could be used to evaluate the participation of essential genes in the mutagenic processes occurring in stressed
cells.
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