Extracellular L-asparaginase production in solid-state fermentation by using sugarcane bagasse as support material.

L-asparaginase is an important enzyme used in the pharmaceutical and food industry, which can be produced by different microorganisms using low cost feedstocks. In this work, sugarcane bagasse (SCB) was used as support for enzyme production in solid-state fermentation (SSF) by A. terreus. Initially, the influence of the variables carbon and nitrogen sources on the enzyme production was studied following an experimental design carried out in Erlenmeyer flasks. Statistical analysis indicated the use of 0.54% of starch, 0% of maltose, 0.44% of asparagine, and 1.14% of glutamine in the medium, resulting in enzyme activity per volume of produced extract of 120.723 U/L. Then, these conditions were applied in a horizontal column reactor filled with SCB, producing 105.3 U/L of enzyme activity. Therefore, the potential of extracellular L-asparaginase enzyme production in the column reactor using sugarcane bagasse as support was demonstrated and it represents a system that can favor large scale production.

About the in vivo quantitation of skin anisotropy.

BACKGROUND/PURPOSE: Human skin anisotropy is difficult to quantify. The Cutiscan® , is allegedly, the first biometrical system to provide information on the elastic and viscoelastic properties, as well as on anisotropy and directionality of the human skin in vivo. Thus, this study aims to contribute to characterize this new device and its applicability, and to compare its behavior with two other well-known devices-the Cutometer® and the Reviscometer® . METHODS: Measurements were conducted with each device in three different anatomical sites (forehead, forearm and leg) of 20 female volunteers engaged after informed consent. The participants in the study were aged 19-73 years (mean age 37 ± 18.7 years old), and were divided in two groups (n = 10), based on their age - Group I, mean age 22 ± 1.3 years; Group II, mean age 52 ± 13.7 years. RESULTS: All devices were useful tools to explore the anatomical and the age dependant changes in biomechanical terms, showing different discriminative capacities. Interesting correlations were established between the variables provided by the equipment. CONCLUSION: The Cutiscan® descriptors delivered excellent relationships with those from Cutometer® and Reviscometer® , while providing more detailed information about skin anisotropy through a full 360° analysis.

Hemicellulosic Ethanol Production by Immobilized Wild Brazilian Yeast Scheffersomyces shehatae UFMG-HM 52.2: Effects of Cell Concentration and Stirring Rate.

The use of sugarcane bagasse hemicellulosic hydrolysates presents an interesting alternative to second generation (2G) ethanol production. Techniques to enhance the fermentation process, e.g., the use of immobilized cells, is one of the key factors for efficient production. Here, the effect of two important parameters (cell concentration in immobilized system and stirring rate) on the 2G ethanol production using the wild Brazilian yeast S. shehatae UFMG-HM 52.2 immobilized in calcium alginate matrix are presented. A 2(2) full factorial design of experiments was carried out to evaluate the effect of cell concentrations in sodium alginate solution for immobilized bead production (3.0, 6.0, and 9.0 g/L) and stirring rate (150, 200, and 250 rpm) for 2G ethanol production. Statistical analysis showed that the use of both variables at low levels enhanced ethanol yield (YP/S). Under these process conditions, YP/S of 0.31 g/g and ethanol productivity (Qp) of 0.12 g/L h were achieved. Results showed the potential of this immobilized yeast in 2G ethanol production from C5 sugars and demonstrate the importance of adequate cell concentration in immobilized systems, a finding that stands to increase bioprocesses yields and productivity.

(1-3)-beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients.

Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1-3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (P ≤ 0.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3 %, 61.3 %, 85.1 %, 79.2 %, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3 %, 35.5 %, 75.9 %, 64.7 %, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400 pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8 % sensitivity, 83.9 % specificity, 92.8 % PPV, 83.9 % NPV, 5.764 PLR and 0.086 NLR (P < 0.001). This study confirmed that BG is a reliable indicator for detecting P. jirovecii infection. The combination between BG/LDH levels and clinical data is a promising alternative approach for PcP diagnosis.

Hydrodynamic cavitation-assisted acid pretreatment and fed-batch simultaneous saccharification and co-fermentation for ethanol production from sugarcane bagasse using immobilized cells of Scheffersomyces parashehatae.

A new alternative for hydrodynamic cavitation-assisted pretreatment of sugarcane bagasse was proposed, along with a simultaneous saccharification and co-fermentation (SSCF) process performed in interconnected columns. Influential variables in the pretreatment were evaluated using a statistical design, indicating that an ozone flow rate of 10 mg min-1 and a pH of 5.10 resulted in 86 % and 72 % glucan and xylan hydrolysis yields, respectively, in the subsequent enzymatic hydrolysis process. Under these optimized conditions, iron sulfate (15 mg L-1) was added to assess Fenton pretreatment, resulting in glucan and xylan hydrolysis yields of 92 % and 71 %, respectively, in a material pretreated for 10 min. In SSCF, ethanol volumetric productivities of 0.33 g L-1 h-1 and of 0.54 g L-1 h-1 were obtained in batch and fed-batch operation modes, achieving 26 g L-1 of ethanol in 48 h in the latter mode.

Stable expression of shRNA for the control of recombinant adenovirus replication.

Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.

Psychosocial factors associated with disability in patients with non-specific chronic low back pain: A cross-sectional study.

INTRODUCTION AND OBJECTIVES: Chronic low back pain is the main cause of disability worldwide, generating high costs for society. To evaluate the prevalence of disability in patients with non-specific chronic low back pain and associated factors, including the impacts of low back pain and psychosocial factors linked to kinesiophobia, catastrophism, anxiety, and depression. PATIENTS: A cross-sectional study was carried out with 108 adult individuals who had non-specific chronic low back pain. The patients answered previously validated questionnaires, namely the Brief Pain Inventory, the Roland-Morris Disability Questionnaire, the Pain Catastrophizing Scale, the Tampa Kinesiophobia Scale, and the Hospital Anxiety and Depression Scale. RESULTS: The prevalence of disability observed was 65.7%, with the mean disability score being 15.7±5.3 points in the Roland-Morris Disability Questionnaire. Although pain intensity and other domains of the Brief Pain Inventory, like anxiety, depression, and severe kinesiophobia were significant in the bivariate analyses, they were not associated with disability in the multivariate analysis. Only catastrophic thoughts (prevalence ratio [PR]=1.19; 95% confidence interval [CI]: 1.07-1.32), and the 'walking' domain (PR=1.08; 95% CI: 1.03-1.14) remained statistically associated with disability. CONCLUSION: Pain catastrophization and impact on gait were associated with disability in individuals with non-specific chronic low back pain. Motor control thoughts and behaviors during functional activities were considered to be relevant aspects for the better assessment and treatment of these patients.

Population mobility and the changing epidemics of HIV-2 in Portugal.

INTRODUCTION: Portugal is the European country with the highest frequency of HIV-2 infection, which is mainly concentrated in West Africa. The cumulative number of notified HIV-2 infections in Portugal was 1813 by the end of December 2008. To better characterize the dynamics of HIV-2 infection in the country and to obtain data that may be of use in the prevention of the spread of HIV-2, we evaluated a large pooled sample of patients. PATIENTS AND METHODS: Five Portuguese hospitals provided data on HIV-2-infected patients from 1984 to the end of 2007. Data concerning demographic characteristics and clinical variables were extracted. Patients were stratified according to date of diagnosis in approximately 5-year categories. RESULTS: The sample included 442 patients, accounting for 37% of all HIV-2 infections notified in Portugal during that period. HIV-2-infected patients showed clearly different characteristics according to the period of diagnosis. Until 2000, the majority of HIV-2-infected patients were Portuguese-born males living in the north of the country. From 2000 to 2007, most of the patients diagnosed with HIV-2 infection had a West African origin, were predominantly female and were living in the capital, Lisbon. The average age at diagnosis and loss to follow-up significantly increased over time. CONCLUSION: HIV-2 infection has been documented in Portugal since the early 1980s and its epidemiology appears to reflect changes in population movement. These include the movements of soldiers and repatriates from African territories during the independence wars and, later, migration and mobility from high-endemicity areas. The findings of this study stress the importance of promoting migrant-sensitive health care.

Toxicity and genotoxicity of organic and inorganic nanoparticles to the bacteria Vibrio fischeri and Salmonella typhimurium.

The present work aimed at evaluating the toxicity and genotoxicity of two organic (vesicles composed of sodium dodecyl sulphate/didodecyl dimethylammonium bromide-SDS/DDAB and of monoolein and sodium oelate-Mo/NaO) and four inorganic (titanium oxide-TiO2, silicon titanium-TiSiO4, Lumidot-CdSe/ZnS, and gold nanorods) nanoparticles (NP), suspended in two aqueous media (Milli Q water and American Society for Testing and Materials (ASTM) hardwater), to the bacteria Vibrio fischeri (Microtox test) and Salmonella typhimurium-his⁻ (Ames test with strains TA98 and TA100). Aiming a better understanding of these biological responses physical and chemical characterization of the studied NP suspensions was carried out. Results denoted a high aggregation state of the NP in the aqueous suspensions, with the exception of SDS/DDAB and Mo/NaO vesicles, and of nanogold suspended in Milli Q water. This higher aggregation was consistent with the low values of zeta potential, revealing the instability of the suspensions. Regarding toxicity data, except for nano TiO2, the tested NP significantly inhibited bioluminescence of V. fischeri. Genotoxic effects were only induced by SDS/DDAB and TiO2 for the strain TA98. A wide range of toxicity responses was observed for the six tested NP, differing by more than 5 orders of magnitude, and suggesting different modes of action of the tested NP.

A review on recent developments in hydrodynamic cavitation and advanced oxidative processes for pretreatment of lignocellulosic materials.

Environmental problems due to utilization of fossil-derived materials for energy and chemical generation has prompted the use of renewable alternative sources, such as lignocellulose biomass (LB). Indeed, the production of biomolecules and biofuels from LB is among the most important current research topics aiming to development a sustainable bioeconomy. Yet, the industrial use of LB is limited by the recalcitrance of biomass, which impairs the hydrolysis of the carbohydrate fractions. Hydrodynamic cavitation (HC) and Advanced Oxidative Processes (AOPs) has been proposed as innovative pretreatment strategies aiming to reduce process time and chemical inputs. Therefore, the underlying mechanisms, procedural strategies, influence on biomass structure, and research gaps were critically discussed in this review. The performed discussion can contribute to future developments, giving a wide overview of the main involved aspects.

Clinical relevance of multiple single-nucleotide polymorphisms in Pneumocystis jirovecii Pneumonia: development of a multiplex PCR-single-base-extension methodology.

Pneumocystis jirovecii pneumonia (PcP) is a major cause of respiratory illness in patients with AIDS. The identification of multiple single-nucleotide polymorphisms (SNPs) at three distinct P. jirovecii loci encoding dihydrofolate reductase (DHFR), mitochondrial large-subunit rRNA (mtLSU rRNA), and superoxide dismutase (SOD) was achieved using multiplex-PCR (MPCR) followed by direct sequencing and two single-base extension (SBE) techniques. Four SNPs (DHFR312, mt85, SOD215, and SOD110), correlated previously with parameters of disease, were amplified and genotyped simultaneously. The concordance of results between the standard sequencing technique (direct sequencing) and SBE analysis was 96.9% for the acrylamide gel electrophoresis and 98.4% for the capillary electrophoresis. The cross-genetic analysis established several statistical associations among the SNPs studied: mt85C-SOD110T, SOD110T-SOD215C, and SOD110C-SOD215T. These results were confirmed by cluster analysis. Data showed that among the isolates with low to moderate parasite burden, the highest percentages of DHFR312C, mt85C, SOD110T, and SOD215C were detected, whereas for high parasite burden cases the highest frequencies were observed among isolates with DHFR312T, mt85T, SOD110C, and SOD215T. The polymorphisms studied were shown to be suitable genetic targets potentially correlated with PcP clinical data that can be used as predictors of outcome in further studies to help clinical decision-making in the management of PcP. The MPCR/SBE protocol described for the first time in the present study was shown to be a rapid, highly accurate method for genotyping P. jirovecii SNPs encoded by different loci that could be used for epidemiological studies and as an additional procedure for the prognostic classification and diagnosis of PcP.

Antibacterial activity of two MTA-based root canal sealers.

AIM: To evaluate the pH and antibacterial activity of Endo CPM Sealer and MTA Fillapex by two different methods, using white MTA and Endofill as references for comparison. METHODOLOGY: Antibacterial activity was evaluated against Enterococcus faecalis (ATCC 29212). The agar diffusion test (ADT) was performed to evaluate the effect before setting. The materials were placed in four equidistant wells made in ten agar plates. After incubation at 37 °C for 48 h, the inhibition zones were measured using a digital paquimeter. The direct contact test (DCT) was performed to assess the antibacterial effect after setting. Suspensions of crushed materials were prepared and mixed with E. faecalis. After different periods of time (1, 6, 15 and 60 min), the survival of bacteria was assessed by using 10-fold serial dilution and cultivated on agar plates in triplicate. Colony-forming units (CFU) mL(-1) were calculated after incubation. pH values were also measured in triplicate. Comparison between sealers in the ADT and DCT was performed by the Kruskal-Wallis test. RESULTS: In the ADT, inhibition zones were found with MTA Fillapex and Endofill. They were similar to each other and greater than the other sealers (P < 0.05). None of the tested sealers demonstrated antibacterial activity in the DCT, and thus, all sealers had similar bacterial counts compared with the negative control group (P > 0.05). White MTA and Endo CPM Sealer suspensions had pH values >11, whilst MTA Fillapex and Endofill had lower values. CONCLUSIONS: MTA Fillapex and Endofill had an antibacterial effect against E. faecalis before setting, but none of the sealers maintained antibacterial activity after setting, despite the high pH of the MTA-based materials.

Intravenous administration of azumolene to reverse malignant hyperthermia in swine.

BACKGROUND: The efficacy of intravenous (IV) administration of azumolene (Az), an analogue 30-fold more soluble than dantrolene, on pigs susceptible to malignant hyperthermia (MH) is incompletely understood. OBJECTIVE: To evaluate efficacy of Az on MH crisis in pigs. ANIMALS: Eight normal (MHN) and 7 susceptible to MH (MHS) pigs (Landrace × Large White × Pietran). METHODS: Prospective, laboratory trial. Hypermetabolic crisis was observed in MHS pigs, but not in MHN pigs, after a combined administration of inhaled halothane (1.5%) and IV injection of succinylcholine (SCh; 2.5 mg/kg). Susceptibility was confirmed using a caffeine and halothane contracture test. Az was administered 15 minutes after administration of SCh. RESULTS: Respiratory acidosis (pH 7.16 ± 0.02; Pco(2) , 46.2 ± 9.1 mmHg, HCO(3) , 22.5 ± 2.3 mmol/L), fever (38.2 ± 1.1°C), cardiac arrhythmias, and muscle contracture were observed in MHS pigs. MHS pigs (n = 5) treated with Az (2 mg/kg IV) survived the crisis with attenuation of signs (pH 7.30 ± 0.10; Pco(2) , 36.3 ± 4.5 mmHg; HCO(3) , 22.9 ± 2.3 mmol/L) and recovery of normal muscle tone and cardiac rhythm. CONCLUSIONS AND CLINICAL IMPORTANCE: Az represents a possible substitute for dantrolene to reverse MH crisis in susceptible pigs.

Blood cell membrane fluidity and intracellular Ca2+ changes in antiretroviral-naïve and -treated HIV-1-infected patients.

We previously showed that lymphocytes and erythrocytes of HIV-1-infected patients, prior to antiretroviral therapy, presented significant changes in intracellular calcium concentration ([Ca(2+)](int)) and membrane fluidity. The present study evaluates the same parameters after response to highly active antiretroviral therapy (HAART). Blood samples were collected from patients prior to and after antiretroviral therapy, and from control subjects. Membrane fluidity and [Ca(2+)](int) were assessed by fluorescence spectroscopy measurements, using three different probes: TMA-DPH and DPH for membrane fluidity, and fura-2 for Ca(2+). When compared with the control group, both untreated and treated patients presented increased lymphocyte [Ca(2+)](int) and decreased lymphocyte membrane fluidity, without significant differences between the two groups of patients. On the contrary, the therapy reversed the membrane fluidity variations observed in erythrocytes. The decreased erythrocyte [Ca(2+)](int) of untreated patients was not reversed by HAART. AIDS patients present changes in lymphocyte (mostly noninfected) and erythrocyte properties, partially reversed by HAART, consistent with a process of facilitated propagation of the infection to new cells, stimulation of virion production, and maintenance of a reservoir of erythrocyte-bound infectious virus. These observations can be related with the action of the HIV Nef protein in the cell's proteins and lipid composition, as well as with the recently observed cell infection by HIV-1 via endocytosis.

Studying the toxicity of SLEnS-LAS micelles to collembolans and plants: Influence of ethylene oxide units in the head groups.

Mixed micelles of linear alkylbenzene sulfonic acid (LAS) and ether sulfate-based surfactants (SLEnS) can be added in household products and cleaning agents. SLEnS with higher ethylene oxide (EO) units in the head groups have economic and environmental advantages. This work aims to assess the influence of the number of EO units in the ecotoxicity of seven variants of SLEnS-LAS micelles (0-50 EO units) in soils. Ecotoxicological tests were carried out to assess emergence and growth of four plants species and reproduction of collembolans. Most of the variants inhibited plants growth at the highest concentrations (1237.5 µg SLEnS kg-1 of soildw). For reproduction, lower number of EO units resulted in EC50 from 924.2 (95 % CL: 760.7-1063.4) to 963.2 (95 % CL: 676.9-1249.6) µg SLEnS kg-1 of soildw, whereas for higher number of EO units (50 and 30) no inhibition was reported. Based on these results, we suggest that a higher number of EO units contribute to less hazardous formulations, confirming that different designs of surfactants may contribute to changes in the responses of terrestrial organisms. Therefore, we demonstrate that standardized ecotoxicological assays may contribute to more sustainable and effective formulations, when used upstream, prior to manufacture and marketing.

Genetic characterization of the UCS and Kex1 loci of Pneumocystis jirovecii.

Nucleotide variation in the Pneumocystis jirovecii upstream conserved sequence (UCS) and kexin-like serine protease (Kex1) loci was studied in pulmonary specimens from Portuguese HIV-positive patients. DNA was extracted and used for specific molecular sequence analysis. The number of UCS tandem repeats detected in 13 successfully sequenced isolates ranged from three (9 isolates, 69%) to four (4 isolates, 31%). A novel tandem repeat pattern and two novel polymorphisms were detected in the UCS region. For the Kex1 gene, the wild-type (24 isolates, 86%) was the most frequent sequence detected among the 28 sequenced isolates. Nevertheless, a nonsynonymous (1 isolate, 3%) and three synonymous (3 isolates, 11%) polymorphisms were detected and are described here for the first time.

Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal.

AIMS: Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium spp. and Giardia spp. Their presence in aquatic systems makes it essential to develop preventive strategies for water and food safety. This study was undertaken to monitor the presence of Cryptosporidium and Giardia in a total of 175 water samples, including raw and treated water from both surface and ground sources in Portugal. METHODS AND RESULTS: The samples were processed according to USEPA Method 1623 for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts, followed by detection of oocysts/cysts by immunofluorecence (IFA) microscopy, PCR-based techniques were done on all water samples collected. Out of 175 samples, 81 (46.3%) were positive for Cryptosporidium and 67 (38.3%) for Giardia by IFA. Cryptosporidium spp. and G. duodenalis genotypes were identified by PCR in 37 (21.7%) and 9 (5.1%) water samples, respectively. C. parvum was the most common species (78.9%), followed by C. hominis (13.2%), C. andersoni (5.3%), and C. muris (2.6%). Subtype IdA15 was identified in all C. hominis-positive water samples. Subtyping revealed the presence of C. parvum subtypes IIaA15G2R1, IIaA16G2R1 and IIdA17G1. Giardia duodenalis subtype A1 was identified. CONCLUSIONS: The results of the present study suggest that Cryptosporidium spp. and Giardia spp. were widely distributed in source water and treated water in Portugal. Moreover, the results obtained indicate a high occurrence of human-pathogenic Cryptosporidium genotypes and subtypes in raw and treated water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Thus, water can be a potential vehicle in the transmission of cryptosporidiosis, and giardiasis of humans and animals in Portugal.

Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain.

Pneumonia caused by the opportunistic organism Pneumocystis jirovecii is a clinically important infection affecting AIDS and other immunocompromised patients. The present study aimed to compare and characterise the frequency pattern of DNA sequences from the P. jirovecii mitochondrial large-subunit rRNA (mtLSU rRNA) gene, the dihydropteroate synthase (DHPS) gene and the internal transcribed spacer (ITS) regions of the nuclear rRNA operon in specimens from Lisbon (Portugal) and Seville (Spain). Total DNA was extracted and used for specific molecular sequence analysis of the three loci. In both populations, mtLSU rRNA gene analysis revealed an overall prevalence of genotype 1. In the Portuguese population, genotype 2 was the second most common, followed by genotype 3. Inversely, in the Spanish population, genotype 3 was the second most common, followed by genotype 2. The DHPS wild-type sequence was the genotype observed most frequently in both populations, and the DHPS genotype frequency pattern was identical to distribution patterns revealed in other European studies. ITS types showed a significant diversity in both populations because of the high sequence variability in these genomic regions. The most prevalent ITS type in the Portuguese population was Eg, followed by Cg. In contrast to other European studies, Bi was the most common ITS type in the Spanish samples, followed by Eg. A statistically significant association between mtLSU rRNA genotype 1 and ITS type Eg was revealed.

Nutritional shortage augments cisplatin-effects on murine melanoma cells.

Melanoma incidence increases every year worldwide and is responsible for 80% of skin cancer deaths. Due to its metastatic potential and resistance to almost any treatments such as chemo, radio, immune and targeted-therapy, the patients still have a poor prognosis, especially at metastatic stage. Considering that, it is crucial to find new therapeutic approaches to overcome melanoma resistance. Here we investigated the effect of cisplatin (CDDP), one of the chemotherapeutic agents used for melanoma treatment, in association with nutritional deprivation in murine melanoma cell lines. Cell death and autophagy were evaluated after the treatment with cisplatin, nutritional deprivation and its association using an in vitro model of murine melanocytes malignant transformation to metastatic melanoma. Our results showed that nutritional deprivation augmented cell death induced by cisplatin in melanoma cells, especially at the metastatic subtype, with slight effects on melanocytes. Mechanistic studies revealed that although autophagy was present at high levels in basal conditions in melanoma cells, was not essential for cell death process that involved mitochondrial damage, reactive oxygen species production and possible glycolysis inhibition. In conclusion, nutritional shortage in combination with chemotherapeutic drugs as cisplatin can be a valuable new therapeutic strategy to overcome melanoma resistance.

Stable expression of shRNA for the control of recombinant adenovirus replication

Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.