Galactose-1-phosphate inhibits cytochrome c oxidase and causes mitochondrial dysfunction in classic galactosemia.

Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid - that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia.

The complete mitochondrial genome of Sporophila nigricollis (Aves, Passeriformes).

BACKGROUND: Sporophila nigricollis, popularly known as Yellow-bellied Seedeater, is a commonly trafficked bird in Brazil. This study describes the complete mitogenome of the species and its phylogenetic position. METHODS AND RESULTS: DNA sample was sequenced on MiSeq (Illumina®) sequencer. Reads were assembled to reference using Geneious. A total of 11,740 paired sequence reads were used to produce the complete mitochondrial DNA sequence with average coverage of 176x. The mitogenome was found to be circular in shape and consisted of 16,777 base pairs. The mitochondrial genome was annotated with MITOS and verified with ExPASy. Its structure contains two ribosomal RNA (rRNA) genes, 22 transporter RNA (tRNA) genes, 13 protein-coding genes (PCGs) and one control region. Twelve of the 13 PCGs have ATG as start codon. Seven of the 13 PCGs contain a TAA stop codon. Most of the tRNA genes and PCGs are encoded on the heavy strand. Phylogenetic analyses were conducted with MEGA using the maximum likelihood method. Sporophila nigricollis grouped together with other Thraupidae species. CONCLUSION: This study presents the first complete mitogenome of Sporophila nigricollis and can be useful for research on evolution, ecology and conservation of this species.

Sphingolipid depletion suppresses UPR activation and promotes galactose hypersensitivity in yeast models of classic galactosemia.

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations on the GALT gene, which encodes the Leloir pathway enzyme galactose-1-phosphate uridyltransferase. Previous studies have shown that the endoplasmic reticulum unfolded protein response (UPR) is relevant to galactosemia, but the molecular mechanism behind the endoplasmic reticulum stress that triggers this response remains elusive. In the present work, we show that the activation of the UPR in yeast models of galactosemia does not depend on the binding of unfolded proteins to the ER stress sensor protein Ire1p since the protein domain responsible for unfolded protein binding to Ire1p is not necessary for UPR activation. Interestingly, myriocin - an inhibitor of the de novo sphingolipid synthesis pathway - inhibits UPR activation and causes galactose hypersensitivity in these models, indicating that myriocin-mediated sphingolipid depletion impairs yeast adaptation to galactose toxicity. Supporting the interpretation that the effects observed after myriocin treatment were due to a reduction in sphingolipid levels, the addition of phytosphingosine to the culture medium reverses all myriocin effects tested. Surprisingly, constitutively active UPR signaling did not prevent myriocin-induced galactose hypersensitivity suggesting multiple roles for sphingolipids in the adaptation of yeast cells to galactose toxicity. Therefore, we conclude that sphingolipid homeostasis has an important role in UPR activation and cellular adaptation in yeast models of galactosemia, highlighting the possible role of lipid metabolism in the pathophysiology of this disease.

Investigating microcrystalline cellulose crystallinity using Raman spectroscopy.

Microcrystalline cellulose (MCC) is a semi-crystalline material with inherent variable crystallinity due to raw material source and variable manufacturing conditions. MCC crystallinity variability can result in downstream process variability. The aim of this study was to develop models to determine MCC crystallinity index (%CI) from Raman spectra of 30 commercial batches using Raman probes with spot sizes of 100 µm (MR probe) and 6 mm (PhAT probe). A principal component analysis model separated Raman spectra of the same samples captured using the different probes. The %CI was determined using a previously reported univariate model based on the ratio of the peaks at 380 and 1096 cm-1. The univariate model was adjusted for each probe. The %CI was also predicted from spectral data from each probe using partial least squares regression models (where Raman spectra and univariate %CI were the dependent and independent variables, respectively). Both models showed adequate predictive power. For these models a general reference amorphous spectrum was proposed for each instrument. The development of the PLS model substantially reduced the analysis time as it eliminates the need for spectral deconvolution. A web application containing all the models was developed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10570-021-04093-1.

Determination of co-crystal phase purity by mid infrared spectroscopy and multiple curve resolution.

Multivariate Curve Resolution (MCR) was used to determine the phase purity of pharmaceutical co-crystals from mid infrared spectra. An in-silico coformer screening was used to choose one of ten potential coformers. This analysis used quantum chemistry simulation to predict which coformers are thermodynamically inclined to form cocrystals with the model drug, hydrochlorothiazide. The coformer chosen was nicotinamide. An experimental solvent screening by ultrasound assisted slurry co-crystallization was performed to evaluate the capacity of the method to determine phase purity. Afterwards, slurry and slow evaporation co-crystallizations were performed at 10, 25, and 40 °C using 7 solvent systems, and two levels of agitation for the evaporation co-crystallization (on and off). Mid infrared spectroscopy (MIRS) analysis of the products of these co-crystallizations was used to develop an MCR model to determine co-crystal phase purity. The MCR results were compared with a reference co-crystal. Experimental design (DoE) was used to investigate the effect of solvents, temperature, and agitation on the purity of co-crystals produced by slurry and evaporation co-crystallization. DoE revealed that evaporation co-crystallization with agitating at 65 rpm formed co-crystals with greater phase purity. The optimal temperature varied with the solvent used.

Application of percolation threshold to disintegration and dissolution of ibuprofen tablets with different microcrystalline cellulose grades.

The study presented was conducted to determine whether a percolation threshold value, previously determined for ibuprofen/microcrystalline cellulose (MCC) blends using percolation theory and compression data (Queiroz et al., 2019), could translate to tablet disintegration and dissolution data. The influence of MCC grade (air stream dried versus spray dried) on tablet disintegration and dissolution was also investigated. Complementary to conventional disintegration and dissolution testing, Raman imaging determined drug distribution within tablets, and in-line particle video microscopy (PVM) and focused-beam reflectance measurement (FBRM) monitored tablet disintegration. Tablets were prepared containing 0-30% w/w ibuprofen. Raman imaging confirmed the percolation threshold by quantifying the number and equivalent circular diameters of ibuprofen domains on tablet surfaces. Across the percolation threshold, a step change in dissolution behaviour occurred, and tablets containing air stream dried MCC showed slower disintegration rates compared to tablets containing spray dried MCC. Dissolution measurements confirmed experimentally a percolation threshold in agreement with that determined using percolation theory and compression data. An increase in drug domains, due to cluster formation, and less efficient tablet disintegration contributed to slower ibuprofen dissolution above the percolation threshold. Slower dissolution was measured for tablets containing air stream dried compared to spray dried MCC.

Diversity of Metal-Resistant and Tensoactive-Producing Culturable Heterotrophic Bacteria Isolated from a Copper Mine in Brazilian Amazonia.

Bacterial extracellular polymeric substances (EPSs) present diverse properties of biotechnological interest, such as surface modification, metal adsorption and hydrophobic substances solubilization through surface tension reduction. Thus, there is a growing demand for new producing strains and structurally variable biomolecules with different properties. One approach for scanning this biodiversity consists of exploring environments under selective pressures. The aim of this study was to evaluate the composition of culturable heterotrophic bacterial communities from five different sites from a copper mine in the Amazon biome by an enrichment technique to obtain metal resistant bacteria (lead, arsenic, cadmium, copper and zinc) capable of producing EPSs. The bacterial densities at the sites varied from 2.42 × 103 to 1.34 × 108 NMP mL-1 and the 77 bacterial isolates obtained were classified in four divisions, ß-Proteobacteria (16.88%), γ-Proteobacteria (7.29%), Firmicutes (61%) and Actinobacteria (12.98%). Bacillus, Alcaligenes, and Lysinibacillus were the most dominant among the 16 observed genera, but the relative frequency of each varied according to the sample and the metal used in the enrichment culture. 58% of the bacterial strains (45) could produce EPSs. From these, 33 strains showed emulsifying activity (E24), and 9 of them reached values higher than 49%. Only Actinomyces viscosus E3.Pb5 and Bacillus subtilis group E3.As2 reduced the medium surface tension to values lower than 35 mN m-1. It was possible to confirm the high presence of bacteria capable of producing EPSs with tensoactive properties in Amazon copper mines and the evolutionary pressure exerted by the heavy metals during enrichment. These molecules can be tested as an alternative for use in processes that involve the removal of metals, such as the bioremediation of contaminated environments.

The yeast protein Ubx4p contributes to mitochondrial respiration and lithium-galactose-mediated activation of the unfolded protein response.

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia.

Complete mitochondrial genome sequence of Myrmecophaga tridactyla from Brazilian Savanna.

Myrmecophaga tridactyla, popularly known as giant anteater, is a member of Xenarthra magnorder which is under the threat of extinction. Herein, we describe the complete mitochondrial genome of M. tridactyla. The circular DNA molecule is 16,546 bp long, contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a non-coding Control Region of 1110 bp. All protein-coding genes are on the heavy strand, except for Nd6. Ten of the 13 PCGs contained an ATG start codon.

The galactose-induced decrease in phosphate levels leads to toxicity in yeast models of galactosemia.

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations in the GALT gene. A number of evidences indicate that the galactose-1-phosphate accumulation observed in patient cells is a cause of toxicity in this disease. Nevertheless, the consequent molecular events caused by the galactose-1-phosphate accumulation remain elusive. Here we show that intracellular inorganic phosphate levels decreased when yeast models of classic galactosemia were exposed to galactose. The decrease in phosphate levels is probably due to the trapping of phosphate in the accumulated galactose-1-phosphate since the deletion of the galactokinase encoding gene GAL1 suppressed this phenotype. Galactose-induced phosphate depletion caused an increase in glycogen content, an expected result since glycogen breakdown by the enzyme glycogen phosphorylase is dependent on inorganic phosphate. Accordingly, an increase in intracellular phosphate levels suppressed the galactose effect on glycogen content and conferred galactose tolerance to yeast models of galactosemia. These results support the hypothesis that the galactose-induced decrease in phosphate levels leads to toxicity in galactosemia and opens new possibilities for the development of better treatments for this disease.

Complete mitochondrial genome of Sporophila maximiliani (Ave, Passeriformes).

Sporophila maximiliani, commonly known as Great-billed Seed-Finch or 'bicudo', is a trafficked bird in Brazil due to the species' beauty and singing, which is appreciated by breeders and collectors. Generally, the Environmental Military Police and IBAMA maintain enforcement actions, rescue work, and seizure of illegally traded of 'bicudo' specimens. The genomic DNA of one specimen was sequenced on MiSeq (Illumina) sequencer. The reads obtained were analyzed, trimmed, and de novo assembled using CLC Workbench® v9.0 (CLC Bio-Qiagen). The mitochondrial genome of S. maximiliani consisted of 16,765 base pairs, 2 ribosomal RNA, 22 transporter RNA, 13 protein-coding genes, and 1 control region. The molecular phylogeny demonstrated that the mitochondrial genome of S. maximiliani diverged from others related Passeriformes.

Dengue virus requires the CC-chemokine receptor CCR5 for replication and infection development.

Dengue is a mosquito-borne disease that affects millions of people worldwide yearly. Currently, there is no vaccine or specific treatment available. Further investigation on dengue pathogenesis is required to better understand the disease and to identify potential therapeutic targets. The chemokine system has been implicated in dengue pathogenesis, although the specific role of chemokines and their receptors remains elusive. Here we describe the role of the CC-chemokine receptor CCR5 in Dengue virus (DENV-2) infection. In vitro experiments showed that CCR5 is a host factor required for DENV-2 replication in human and mouse macrophages. DENV-2 infection induces the expression of CCR5 ligands. Incubation with an antagonist prevents CCR5 activation and reduces DENV-2 positive-stranded (+) RNA inside macrophages. Using an immunocompetent mouse model of DENV-2 infection we found that CCR5(-/-) mice were resistant to lethal infection, presenting at least 100-fold reduction of viral load in target organs and significant reduction in disease severity. This phenotype was reproduced in wild-type mice treated with CCR5-blocking compounds. Therefore, CCR5 is a host factor required for DENV-2 replication and disease development. Targeting CCR5 might represent a therapeutic strategy for dengue fever. These data bring new insights on the association between viral infections and the chemokine receptor CCR5.

Altered intracellular signaling cascades in peripheral blood mononuclear cells from BD patients.

Bipolar disorder (BD) is a severe psychiatric disorder of complex physiopathology that has been associated with a pro-inflammatory state. The aim of the present study was to investigate intracellular pathways associated with inflammatory signaling, assessing the phosphorylation levels of transcription factor nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPKs) in peripheral blood mononuclear cells of euthymic BD patients and healthy controls. Fifteen BD euthymic type I patients, and 12 healthy controls matched by age and gender were enrolled in this study. All subjects were assessed by the Mini-International Neuropsychiatry Interview and the patients also by the Young Mania Rating Scale and the Hamilton Depression Rating Scale. Phosphorylation levels of p65 NF-κB subunit, and MAPK ERK1/2, and p38 were assessed by Western blot and flow cytometry. Plasma cytokines (IL-2, IL-4, IL6, IL-10, IFN-γ, TNF-α, and IL-17A) were measured using cytometric bead arrays. Western blot and flow cytometry analyses showed increased phosphorylation levels of p65 NF-κB subunit, and MAPKs ERK1/2, and p38 in BD patients in euthymia in comparison with controls. BD patients presented increased pro-inflammatory cytokines levels in comparison with controls, and TNF-α correlated with the levels of phosphorylated p65 NF-κB. The present study found increased activation of MAPK and NF-κB pathways in BD patients, which is in line with a pro-inflammatory status.