Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196.

The Aspergillus niger CexA transporter belongs to the DHA1 (Drug-H+ antiporter) family. CexA homologs are exclusively found in eukaryotic genomes, and CexA is the sole citrate exporter to have been functionally characterized in this family so far. In the present work, we expressed CexA in Saccharomyces cerevisiae, demonstrating its ability to bind isocitric acid, and import citrate at pH 5.5 with low affinity. Citrate uptake was independent of the proton motive force and compatible with a facilitated diffusion mechanism. To unravel the structural features of this transporter, we then targeted 21 CexA residues for site-directed mutagenesis. Residues were identified by a combination of amino acid residue conservation among the DHA1 family, 3D structure prediction, and substrate molecular docking analysis. S. cerevisiae cells expressing this library of CexA mutant alleles were evaluated for their capacity to grow on carboxylic acid-containing media and transport of radiolabeled citrate. We also determined protein subcellular localization by GFP tagging, with seven amino acid substitutions affecting CexA protein expression at the plasma membrane. The substitutions P200A, Y307A, S315A, and R461A displayed loss-of-function phenotypes. The majority of the substitutions affected citrate binding and translocation. The S75 residue had no impact on citrate export but affected its import, as the substitution for alanine increased the affinity of the transporter for citrate. Conversely, expression of CexA mutant alleles in the Yarrowia lipolytica cex1Δ strain revealed the involvement of R192 and Q196 residues in citrate export. Globally, we uncovered a set of relevant amino acid residues involved in CexA expression, export capacity and import affinity.

Cytoskeleton disruption by the metabolic inhibitor 3-bromopyruvate: implications in cancer therapy.

The small molecule 3-bromopyruvate (3BP), is an anticancer molecule that acts by hindering glycolysis and mitochondrial function leading to energy depletion and consequently, to cell death. In this work we have focused on understanding how the glycolytic inhibition affects cancer cell structural features. We showed that 3BP leads to a drastic decrease in the levels of ß-actin and α-tubulin followed by disorganization and shrinkage of the cytoskeleton in breast cancer cells. 3BP inhibits cell migration and colony formation independently of the activity of metalloproteinases. To disclose if these structural alterations occurred prior to 3BP toxic effect, non-toxic concentrations of 3BP were used and we could observe that 3BP was able to inhibit energy production and induce loss of ß-actin and α-tubulin proteins. This was accompanied with alterations in cytoskeleton organization and an increase in E-cadherin levels which may indicate a decrease in cancer cells aggressiveness. In this study we demonstrate that 3BP glycolytic inhibition of breast cancer cells is accompanied by cytoskeleton disruption and consequently loss of migration ability, suggesting that 3BP can potentially be explored for metastatic breast cancer therapy.

Permeability and Porosity Development during the Carbonization of Coals of Different Coking Pressures.

To obtain a better understanding of the development of coking pressure during the carbonization process, the plastic and semicoke layers of nine coking coals were investigated. The permeability of the plastic layer to the passage of gas and the porosity of the semicoke were analyzed at two temperatures, 500 and 800 °C. In the case of dangerous coals, there was a wide zone of low permeability covering most of the plastic layer and part of the semicoke, whereas safe coals had a very narrow permeability zone that affected only a small part of the plastic layer. It seems that dangerous coals have a higher porosity and a lower Hg apparent density than safe coals. In addition, the semicokes obtained at 800 °C from the dangerous coals had a higher macropore volume with pore size between 50 nm and 12 µm but a lower suprapore volume (pore size between 12 and 250 µm).

Mortality-related factors in patients with OXA-48 carbapenemase-producing Klebsiella pneumoniae bacteremia.

ABSTRACT: Carbapenemase-producing Enterobacterales constitute a serious public health threat; however, information on the oxacilinasa (OXA-48)-type is limited. The objective of the study was to evaluate the risk factors associated with 14-day mortality for patients with bacteremia due to OXA-48 carbapenemase-producing Klebsiella pneumoniae.We conducted a retrospective, single-center observational study of adult patients with K. pneumoniae bacteremia, classifying the strains as carbapenem-susceptible K. pneumoniae (CSKp) and carbapenem-resistant K. pneumoniae (CRKp). All of the CRKp strains were the OXA-48-type.The study included 202 cases of bacteremia: 114 due to CSKp and 88 due to CRKp. The clinical cure rate was higher for the patients with CSKp (85% vs 69% for CSKp and CRKp, respectively; P = .010), while the 14-day mortality rate was lower (13% vs 30%, P = .005). An INCREMENT-CPE score ≥7 (HR 3.05, 95% CI 1.50-6.25, P = .002) was the only independent factor associated with 14-day mortality for the patients with Klebsiella spp. bacteremia. Other factors related to 14-day mortality were a rapidly fatal prognosis (McCabe) (HR 7.1, 95% CI 2.75-18.37, P < .001), dementia (HR 5.9, 95% CI 2.0-7.43, P = .001), and a high-risk source of infection (HR 2.7, 95% CI 1.06-6.82, P = .038).The most important factors associated with 14-day mortality for the patients with K. pneumoniae bacteremia was an INCREMENT-CPE score ≥7, dementia, a McCabe score indicating a rapidly fatal prognosis and a high-risk source of infection. We found no relationship between a poorer outcome and CRKp isolation or inadequate antibiotic therapy.

Membrane transporters in the bioproduction of organic acids: state of the art and future perspectives for industrial applications.

Organic acids such as monocarboxylic acids, dicarboxylic acids or even more complex molecules such as sugar acids, have displayed great applicability in the industry as these compounds are used as platform chemicals for polymer, food, agricultural and pharmaceutical sectors. Chemical synthesis of these compounds from petroleum derivatives is currently their major source of production. However, increasing environmental concerns have prompted the production of organic acids by microorganisms. The current trend is the exploitation of industrial biowastes to sustain microbial cell growth and valorize biomass conversion into organic acids. One of the major bottlenecks for the efficient and cost-effective bioproduction is the export of organic acids through the microbial plasma membrane. Membrane transporter proteins are crucial elements for the optimization of substrate import and final product export. Several transporters have been expressed in organic acid-producing species, resulting in increased final product titers in the extracellular medium and higher productivity levels. In this review, the state of the art of plasma membrane transport of organic acids is presented, along with the implications for industrial biotechnology.

Production of bioactive hepcidin by recombinant DNA tagging with an elastin-like recombinamer.

With the lack of new chemical antibiotics and increasing pathogen resistance to those available, new alternatives are being explored. Antimicrobial peptides (AMPs) with a broad range of effects, including antibacterial, antifungal, and antiviral actions, have emerged as one of the options. They can be produced by recombinant DNA technology, but the chromatographic methods used for peptide purification are expensive and time consuming. Here, we describe the design, production, purification and assessment of the antibacterial activity of the human peptide hepcidin, using an elastin-like recombinamer as fusion partner. The recombinant protein Hep-A200 was produced in Escherichia coli and purified by a non-chromatographic procedure, exploiting the thermal properties of the A200 elastin-like recombinamer. Recombinant Hep-A200 was found to retain antibacterial activity against Gram-positive and Gram-negative species.

In vitro studies of DNA damage and repair mechanisms induced by BNCT in a poorly differentiated thyroid carcinoma cell line.

Boron neutron capture therapy (BNCT) for aggressive tumors is based on nuclear reaction [10B (n, α) 7Li]. Previously, we demonstrated that BNCT could be applied for the treatment of undifferentiated thyroid carcinoma. The aim of the present study was to describe the DNA damage pattern and the repair pathways that are activated by BNCT in thyroid cells. We analyzed γH2AX foci and the expression of Ku70, Rad51 and Rad54, main effector enzymes of non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, respectively, in thyroid follicular carcinoma cells. The studied groups were: (1) C [no irradiation], (2) gamma [60Co source], (3) N [neutron beam alone], (4) BNCT [neutron beam plus 10 µg 10B/ml of boronphenylalanine (10BPA)]. The total absorbed dose was always 3 Gy. The results showed that the number of nuclear γH2AX foci was higher in the gamma group than in the N and BNCT groups (30 min-24 h) (p < 0.001). However, the focus size was significantly larger in BNCT compared to other groups (p < 0.01). The analysis of repair enzymes showed a significant increase in Rad51 and Rad54 mRNA at 4 and 6 h, respectively; in both N and BNCT groups and the expression of Ku70 did not show significant differences between groups. These findings are consistent with an activation of HRR mechanism in thyroid cells. A melanoma cell line showed different DNA damage pattern and activation of both repair pathways. These results will allow us to evaluate different blocking points, to potentiate the damage induced by BNCT.

Canine GM2-Gangliosidosis Sandhoff Disease Associated with a 3-Base Pair Deletion in the HEXB Gene.

BACKGROUND: GM2-gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either ß-hexosaminidase A (Hex-A) and ß-hexosaminidase B (Hex-B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency. OBJECTIVES: To characterize the phenotype and genotype of GM2-gangliosidosis disease in an affected dog. ANIMALS: One affected Shiba Inu and a clinically healthy dog. METHODS: Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes. RESULTS: A 14-month-old, female Shiba Inu presented with clinical signs resembling GM2-gangliosidosis in humans and GM1-gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex-A and Hex-B activities in both tissues. Genetic analysis identified a homozygous, 3-base pair deletion in the HEXB gene (c.618-620delCCT). CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2-gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2-gangliosidosis seen in this dog is the Sandhoff type. Because GM1-gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1- and GM2-gangliosidosis should be considered to make a definitive diagnosis.

Photon iso-effective dose for cancer treatment with mixed field radiation based on dose-response assessment from human and an animal model: clinical application to boron neutron capture therapy for head and neck cancer.

Boron neutron capture therapy (BNCT) is a treatment modality that combines different radiation qualities. Since the severity of biological damage following irradiation depends on the radiation type, a quantity different from absorbed dose is required to explain the effects observed in the clinical BNCT in terms of outcome compared with conventional photon radiation therapy. A new approach for calculating photon iso-effective doses in BNCT was introduced previously. The present work extends this model to include information from dose-response assessments in animal models and humans. Parameters of the model were determined for tumour and precancerous tissue using dose-response curves obtained from BNCT and photon studies performed in the hamster cheek pouch in vivo models of oral cancer and/or pre-cancer, and from head and neck cancer radiotherapy data with photons. To this end, suitable expressions of the dose-limiting Normal Tissue Complication and Tumour Control Probabilities for the reference radiation and for the mixed field BNCT radiation were developed. Pearson's correlation coefficients and p-values showed that TCP and NTCP models agreed with experimental data (with r > 0.87 and p-values >0.57). The photon iso-effective dose model was applied retrospectively to evaluate the dosimetry in tumours and mucosa for head and neck cancer patients treated with BNCT in Finland. Photon iso-effective doses in tumour were lower than those obtained with the standard RBE-weighted model (between 10% to 45%). The results also suggested that the probabilities of tumour control derived from photon iso-effective doses are more adequate to explain the clinical responses than those obtained with the RBE-weighted values. The dosimetry in the mucosa revealed that the photon iso-effective doses were about 30% to 50% higher than the corresponding RBE-weighted values. While the RBE-weighted doses are unable to predict mucosa toxicity, predictions based on the proposed model are compatible with the observed clinical outcome. The extension of the photon iso-effective dose model has allowed, for the first time, the determination of the photon iso-effective dose for unacceptable complications in the dose-limiting normal tissue. Finally, the formalism developed in this work to compute photon-equivalent doses can be applied to other therapies that combine mixed radiation fields, such as hadron therapy.

Single step fabrication of antimicrobial fibre mats from a bioengineered protein-based polymer.

Genetically engineered protein polymers functionalized with bioactive domains have potential as multifunctional versatile materials for biomedical use. The present work describes the fabrication and characterisation of antimicrobial fibre mats comprising the antimicrobial elastin-like recombinamer (ELR) CM4-A200. The CM4-A200 protein polymer derives from the genetic fusion of the ABP-CM4 antimicrobial peptide from Bombyx mori with 200 repetitions of the pentamer VPAVG. This is the first report on non-crosslinked fibre mats fabricated with an antimicrobial ELR stable in solution. Thermal gravimetric analysis of CM4-A200 fibre mats shows one single degradation step at temperatures above 300 °C, with fibres displaying a higher thermal degradation activation. The electrospun CM4-A200 fibres display high antimicrobial activity against Gram-positive and Gram-negative bacteria with no detectable cytotoxic effects against normal human skin fibroblasts and keratinocytes, revealing the great potential of these polymers for the fabrication of biomedical materials.

The anticancer agent 3-bromopyruvate: a simple but powerful molecule taken from the lab to the bedside.

At the beginning of the twenty-first century, 3-bromopyruvate (3BP), a simple alkylating chemical compound was presented to the scientific community as a potent anticancer agent, able to cause rapid toxicity to cancer cells without bystander effects on normal tissues. The altered metabolism of cancers, an essential hallmark for their progression, also became their Achilles heel by facilitating 3BP's selective entry and specific targeting. Treatment with 3BP has been administered in several cancer type models both in vitro and in vivo, either alone or in combination with other anticancer therapeutic approaches. These studies clearly demonstrate 3BP's broad action against multiple cancer types. Clinical trials using 3BP are needed to further support its anticancer efficacy against multiple cancer types thus making it available to more than 30 million patients living with cancer worldwide. This review discusses current knowledge about 3BP related to cancer and discusses also the possibility of its use in future clinical applications as it relates to safety and treatment issues.

New Anyplex™ II MTB/MDR/XDR kit for detection of resistance mutations in M. tuberculosis cultures.

SETTINGS: The new Anyplex™ II MTB/MDR/XDR PCR assay enables the joint analysis of mutations conferring resistance to first- and second-line anti-tuberculosis drugs and the detection of Mycobacterium tuberculosis. OBJECTIVES: To evaluate the performance of the new Anyplex assay in detecting mutations that confer resistance to first- and second-line drugs in M. tuberculosis cultures. DESIGN: Results obtained using the new technique were compared with those obtained by phenotypic drug susceptibility testing (DST) and with two GenoType tests for the detection of mutations: GenoType(®) MTBDRplus and GenoType(®) MTBDRsl. RESULTS: For rifampicin resistance mutations, Anyplex displayed 97% sensitivity and 100% specificity compared with 100% and 100% for MTBDRplus. For isoniazid (INH) resistance, Anyplex displayed 61% sensitivity and 98% specificity compared with 62% and 98% for MTBDRplus. For second-line drugs, Anyplex recorded 95% sensitivity and 99% specificity in the detection of resistance to quinolones compared with 100% and 98% for the MTBDRsl. While both techniques displayed 100% specificity for aminoglycoside resistance mutations, sensitivity was 100% for Anyplex and 88% for MTBDRsl. CONCLUSIONS: Results obtained using Anyplex agreed strongly with those obtained using the two GenoType molecular techniques and with phenotypic DST, except in the case of INH, due to the large number of genes involved in resistance to this drug.

Treatment guidelines for Hepatitis C in Spain.

The discovery of new orally administered drugs that can block different targets of the replication cycle of the hepatitis C virus (HCV) with major antiviral activity, has revolutionized treatment of this infection and relegated interferon-based treatments to a secondary position. The start up of the National Strategic Plan for Combating Hepatitis C, which acknowledges the greater efficacy and safety of oral antiviral drugs, as well as the agreements between the pharmaceutical companies and different government bodies has enabled the initial difficulties of access to these medicines due to their high cost to be overcome. In this rapidly changing environment, the availability of a therapeutic guide based on a critical analysis of the available evidence, takes on special relevance and provides a basic support for medical practitioners involved in HCV treatment. However, the speed with which new therapeutic options are included and the limited evidence in some clinical scenarios signifies a challenge for those responsible for scientific societies whose job it is to coordinate the preparation of therapeutic guides and to keep recommendations up to date. In this review we analyze the treatment recommendations for HCV in a consensus document drawn up by the Spanish Association for the Study of Liver Diseases (AEEH), to contrast them with recommendations given by American and European associations that study hepatic diseases.

Deceased-donor Kidney Transplantation: Predictive Factors and Impact on Postoperative Outcome.

Kidney transplantation (KT) is the treatment of choice in end stage renal disease. Patients proposed for KT have multiple comorbidities, which makes KT a challenge. Our aim was to assess predictive factors for postoperative complications in deceased-donor KT. For data statistical analysis, logistic and linear regressions were used. Between 2012 and 2013, 113 KTs were performed in patients with a mean age 49.9 years. The most prevalent etiology was unknown (32.7%). All patients were in kidney replacement therapy (KRT), for an average of 5.7 years. Most had comorbidities before KT (84.1%), the most frequent hypertension (82.3%). Mean ischemia time (IT) was 1056 minutes. Complications occurred in 93.8% of cases. There were reinterventions in 12.4% of cases, and reinterventions in 13.3%. The time in KRT, IT, and ischemic heart disease had predictive power for the length of hospital stay. Diabetes mellitus before KT and IT were predictors for nephrourologic complications; anemia before KT for hematologic complications; and anemia before KT and time in KRT for cardiovascular complications. The morbidity associated with this disease points to the need to identify and improve the patient-dependent variables influencing its outcome, so as to improve short-term success.

Living-donor Kidney Transplantation: Predictive Factors and Impact on Post-transplant Outcome.

Renal transplantation from living donors represents a valuable opportunity for patients with end-stage renal disease due to short- and long-term outcomes. Nevertheless, it requires that a detailed set of conditions be considered for donor and recipient selection, with possible implications arising from these criteria in the post-transplant outcome. The present work aims to study demographic and clinical characteristics of donors and kidney recipients that predict post-transplantation outcomes after living donor kidney transplantation. With this aim, all patients who underwent donor nephrectomy and living donor transplantation between January 2012 and December 2013 were selected. Demographics, medical comorbidities, and postoperative outcomes were transcribed from electronic patient records. Linear and logistic regressions were applied for data analysis. The study sample consists of 40 patients who underwent living donor kidney transplantation. The presence of peripheral arterial disease and the etiology of end-stage renal disease were the only pretransplant variables that seem to independently predict hospitalization time. Simultaneously, the occurrence of urorenal and infectious complications had a statistically significant correlation with hospitalization time. Additionally, the incidence of cardiovascular complications was correlated with surgical reinterventions at a significant level. The results suggest that careful selection of the donor and the kidney recipient appears to be a prerequisite for a successful transplantation in vivo.

Living-donor and Deceased-donor Renal Transplantation: Differences in Early Outcome--A Single-center Experience.

Living-donor renal transplant (LDRT) yields better long-term outcomes than cadaver-donor renal transplant (CDRT). The aim of the present study was to identify the differences in the early postoperative period between LDRT and CDRT recipients. A retrospective study was conducted including all patients receiving a LDRT and CDRT in this center in 2012 and 2013. A total of 153 recipients were identified (CDRT n = 113, LDRT n = 40). On average, LDRT recipients were younger by 12.7 years (P < .001) and had fewer comorbidities (P < .05). There were no differences in gender or primary kidney disease. Mean time on dialysis, dialytic technique, and ischemia time were different between groups (P < .001, P < .01, P < .001, respectively). On average the length of hospital stay for LDRT recipients was 7 days shorter (P < .001). We found significant differences in the occurrence of early complications (P < .001) and its subtypes, with the exception of neurologic and respiratory complications. There were no differences in reinterventions and readmissions between groups. Recipients' age was an independent risk factor for overall postoperative complications and infectious complications; hypertension before renal transplant and cold ischemia time were predictors for cardiovascular complications; and cold ischemia time also was a predictor of nephrourologic and endocrine complications. CDRT patients had more postoperative complications during hospital stay. The variables identified as predictors of early outcome were different for the 2 groups of patients. Modifiable risk factors for better early outcomes and the impact of immediate complications in long-term graft survival must be investigated.

Autosomal Recessive Congenital Ichthyosis in American Bulldogs Is Associated With NIPAL4 (ICHTHYIN) Deficiency.

A minority of patients with nonsyndromic autosomal recessive congenital ichthyosis (ARCI) display mutations in NIPAL4 (ICHTHYIN). This protein plays a role in epidermal lipid metabolism, although the mechanism is unknown. The study describes a moderate form of ARCI in an extended pedigree of American Bulldogs that is linked to the gene encoding ichthyin. The gross phenotype was manifest as a disheveled pelage shortly after birth, generalized scaling, and adherent brown scale with erythema of the abdominal skin. Pedigree analysis indicated an autosomal recessive mode of inheritance. Ultrastructurally, the epidermis showed discontinuous lipid bilayers, unprocessed lipid within corneocytes, and abnormal lamellar bodies. Linkage analysis, performed by choosing simple sequence repeat markers and single-nucleotide polymorphisms near genes known to cause ACRI, revealed an association with NIPAL4. NIPAL4 was identified and sequenced using standard methods. No mutation was identified within the gene, but affected dogs had a SINE element 5' upstream of exon 1 in a highly conserved region. Of 545 DNA samples from American Bulldogs, 32 dogs (17 females, 15 males) were homozygous for the polymerase chain reaction fragment. All affected dogs were homozygous, with parents heterozygous for the insertion. Immunolabeling revealed an absence of ichthyin in the epidermis. This is the first description of ARCI associated with decreased expression of NIPAL4 in nonhuman species.

Monocarboxylate transporters as targets and mediators in cancer therapy response.

Monocarboxylate transporters (MCTs) belong to a family of transporters, encoded by the SLC16 gene family, which is presently composed by 14 members, but only MCT1 to 4 have been biochemically characterized. They have important functions in healthy tissues, being involved in the transmembrane transport of lactic acid and other monocarboxylic acids in human cells. One of the recently recognized hallmarks of cancer is altered metabolism, with high rates of glucose consumption and consequent lactate production. To maintain this metabolic phenotype, cancer cells upregulate a series of plasma membrane proteins, including MCTs. MCT1 and MCT4, in particular, play a dual role in the maintenance of the metabolic phenotype of tumour cells. On one hand, they facilitate the efflux of lactate and, on the other hand, they contribute to the preservation of the intracellular pH, by co-transporting a proton. Thus, MCTs are attractive targets in cancer therapy, especially in cancers with a hyper-glycolytic and acid-resistant phenotype. Recent evidence demonstrates that MCTs are involved in cancer cell uptake of chemotherapeutic agents, including 3-bromopyruvate. In this way MCTs can act as "Trojan horses", as their elevated expression in cancer cells can mediate the entry of this chemotherapeutic agent into the cells and selectively kill cancer cells. As a result, MCTs will be mediators of chemotherapeutic response, and their expression can be used as a molecular marker to predict response to chemotherapy.

An easy, quick and cheap high-throughput method for yeast DNA extraction from microwell plates.

We herein present a high-throughput and cheap method for yeast DNA isolation in a 96 well microplate. About 1500 yeast isolates can be processed within one working day and final DNA concentrations are suitable for direct application in PCR-based molecular typing methods.

[Investigation of antimicrobial resistance to Enterococcus faecium]. / Investigación de las resistencias a antimicrobianos en Enterococcus faecium.

We performed a antibiotic resistance study on Enterococcus faecium isolated from intrahospitalary and extrahospitalary samples between 2004 and 2010. Three different samples were studied; urine, blood and wound swabs, considering a strain per patient. We included in the study a global amount of 637 E. faecium isolares. We employed semiautomatic system WIDER I for identification and sensitivity testing. We considered susceptibility and resistance criteria recommended by MENSURA group. We found a susceptibility rate of 48.05% to betalactams, 100% to linezolid, and 99.46% to vancomycin. The resistance to aminoglycosides ranged between 41.41 and 73.55%. We obtained 6 isolates resistant to vancomycin one of them from an extrahospitalary strain and five from intrahospitalary strains. It seems that vancomycin resistance should be controlled.