Isolation of acute myeloid leukemia blasts from blood using a microfluidic device.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and associated with poor prognosis. Unfortunately, most of the patients that achieve clinical complete remission after the treatment will ultimately relapse due to the persistence of minimal residual disease (MRD), that is not measurable using conventional technologies in the clinic. Microfluidics is a potential tool to improve the diagnosis by providing early detection of MRD. Herein, different designs of microfluidic devices were developed to promote lateral and vertical mixing of cells in microchannels to increase the contact area of the cells of interest with the inner surface of the device. Possible interactions between the cells and the surface were studied using fluid simulations. For the isolation of leukemic blasts, a positive selection strategy was used, targeting the cells of interest using a panel of specific biomarkers expressed in immature and aberrant blasts. Finally, once the optimisation was complete, the best conditions were used to process patient samples for downstream analysis and benchmarking, including phenotypic and genetic characterisation. The potential of these microfluidic devices to isolate and detect AML blasts may be exploited for the monitoring of AML patients at different stages of the disease.

Development of Smart Clothing to Prevent Pressure Injuries in Bedridden Persons and/or with Severely Impaired Mobility: 4NoPressure Research Protocol.

Pressure injuries (PIs) are a major public health problem and can be used as quality-of-care indicators. An incipient development in the field of medical devices takes the form of Smart Health Textiles, which can possess innovative properties such as thermoregulation, sensing, and antibacterial control. This protocol aims to describe the process for the development of a new type of smart clothing for individuals with reduced mobility and/or who are bedridden in order to prevent PIs. This paper's main purpose is to present the eight phases of the project, each consisting of tasks in specific phases: (i) product and process requirements and specifications; (ii and iii) study of the fibrous structure technology, textiles, and design; (iv and v) investigation of the sensor technology with respect to pressure, temperature, humidity, and bioactive properties; (vi and vii) production layout and adaptations in the manufacturing process; (viii) clinical trial. This project will introduce a new structural system and design for smart clothing to prevent PIs. New materials and architectures will be studied that provide better pressure relief, thermo-physiological control of the cutaneous microclimate, and personalisation of care.

Advances in Microfluidics for the Implementation of Liquid Biopsy in Clinical Routine.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

Uncovering Novel Plasma Membrane Carboxylate Transporters in the Yeast Cyberlindnera jadinii.

The yeast Cyberlindnera jadinii has great potential in the biotechnology industry due to its ability to produce a variety of compounds of interest, including carboxylic acids. In this work, we identified genes encoding carboxylate transporters from this yeast species. The functional characterization of sixteen plasma membrane carboxylate transporters belonging to the AceTr, SHS, TDT, MCT, SSS, and DASS families was performed by heterologous expression in Saccharomyces cerevisiae. The newly identified C. jadinii transporters present specificity for mono-, di-, and tricarboxylates. The transporters CjAto5, CjJen6, CjSlc5, and CjSlc13-1 display the broadest substrate specificity; CjAto2 accepts mono- and dicarboxylates; and CjAto1,3,4, CjJen1-5, CjSlc16, and CjSlc13-2 are specific for monocarboxylic acids. A detailed characterization of these transporters, including phylogenetic reconstruction, 3D structure prediction, and molecular docking analysis is presented here. The properties presented by these transporters make them interesting targets to be explored as organic acid exporters in microbial cell factories.

Evolutionary engineering reveals amino acid substitutions in Ato2 and Ato3 that allow improved growth of Saccharomyces cerevisiae on lactic acid.

In Saccharomyces cerevisiae, the complete set of proteins involved in transport of lactic acid across the cell membrane has not been determined. In this study, we aimed to identify transport proteins not previously described to be involved in lactic acid transport via a combination of directed evolution, whole-genome resequencing and reverse engineering. Evolution of a strain lacking all known lactic acid transporters on lactate led to the discovery of mutated Ato2 and Ato3 as two novel lactic acid transport proteins. When compared to previously identified S. cerevisiae genes involved in lactic acid transport, expression of ATO3T284C was able to facilitate the highest growth rate (0.15 ± 0.01 h-1) on this carbon source. A comparison between (evolved) sequences and 3D models of the transport proteins showed that most of the identified mutations resulted in a widening of the narrowest hydrophobic constriction of the anion channel. We hypothesize that this observation, sometimes in combination with an increased binding affinity of lactic acid to the sites adjacent to this constriction, are responsible for the improved lactic acid transport in the evolved proteins.

Expanding the Knowledge on the Skillful Yeast Cyberlindnera jadinii.

Cyberlindnera jadinii is widely used as a source of single-cell protein and is known for its ability to synthesize a great variety of valuable compounds for the food and pharmaceutical industries. Its capacity to produce compounds such as food additives, supplements, and organic acids, among other fine chemicals, has turned it into an attractive microorganism in the biotechnology field. In this review, we performed a robust phylogenetic analysis using the core proteome of C. jadinii and other fungal species, from Asco- to Basidiomycota, to elucidate the evolutionary roots of this species. In addition, we report the evolution of this species nomenclature over-time and the existence of a teleomorph (C. jadinii) and anamorph state (Candida utilis) and summarize the current nomenclature of most common strains. Finally, we highlight relevant traits of its physiology, the solute membrane transporters so far characterized, as well as the molecular tools currently available for its genomic manipulation. The emerging applications of this yeast reinforce its potential in the white biotechnology sector. Nonetheless, it is necessary to expand the knowledge on its metabolism, regulatory networks, and transport mechanisms, as well as to develop more robust genetic manipulation systems and synthetic biology tools to promote the full exploitation of C. jadinii.

Carboxylic Acid Transporters in Candida Pathogenesis.

Opportunistic pathogens such as Candida species can use carboxylic acids, like acetate and lactate, to survive and successfully thrive in different environmental niches. These nonfermentable substrates are frequently the major carbon sources present in certain human body sites, and their efficient uptake by regulated plasma membrane transporters plays a critical role in such nutrient-limited conditions. Here, we cover the physiology and regulation of these proteins and their potential role in Candida virulence. This review also presents an evolutionary analysis of orthologues of the Saccharomyces cerevisiae Jen1 lactate and Ady2 acetate transporters, including a phylogenetic analysis of 101 putative carboxylate transporters in twelve medically relevant Candida species. These proteins are assigned to distinct clades according to their amino acid sequence homology and represent the major carboxylic acid uptake systems in yeast. While Jen transporters belong to the sialate:H+ symporter (SHS) family, the Ady2 homologue members are assigned to the acetate uptake transporter (AceTr) family. Here, we reclassify the later members as ATO (acetate transporter ortholog). The new nomenclature will facilitate the study of these transporters, as well as the analysis of their relevance for Candida pathogenesis.

Caracteristicas clinicas y epidemiologicas de lesiones cervicales no cariosas / Clinical and epidemiological characteristics of non-carious cervical injuries / Características clínicas y epidemiológicas de lesões cervicais não cariosas

Las lesiones cervicales no cariosas consisten en la pérdida de estructura dentaria en la región cervical de la superficie vestibular de los elementos dentarios, no relacionados con caries. Objetivo: Verificar las características clínicas, epi-demiológicas y los factores de riesgo de lesiones cervicales no cariosas. Materiales y métodos: Fueron evaluados 300 elementos dentarios en 96 individuos adultos que fueron sometidos a entrevista y examen clínico intra-oral. Resultados: Se encontró una razón de prevalencia en la que 0,87 de los pacientes usan prótesis. Respecto a hábitos de higiene oral, 0,51 utilizan cepillo suave; 0,52 se cepillan tres veces por día y, 0,68 utilizan dirección de cepillado combinado. En los pacientes, se evaluó que 0,56 tenían antecedentes de trastornos gastrointestinales; el 40% toman medicamentos; 0,36 presentan desoclusión en guía canina. Con relación a las características de la lesión, 0,40 presentan tipo de contacto A; 43% con profundidad inferior a 1 mm; 0,13 son portadores de prótesis parcial removible. En cuanto a hábitos alimenticios 0,93 toman bebidas cítricas; 0,15 son fumadores. Referente a los hábitos parafuncionales, 0,35 aprietan los dientes; 0,36 son bruxistas; 0,10 muerden objetos. Estas lesiones presentan una mayor prevalencia en premolares 0,54. Conclusiones: Verificando las características clínicas y epidemiológicas, y los factores de riesgo de las lesiones cervicales no cariosas, se concluye que predominan en premolares, el factor determinante para el surgimiento de estas lesiones es multifactorial, predominando la inestabilidad oclusal, ingestión de bebidas cítricas, uso de prótesis, trastornos gastrointestinales y hábitos parafuncionales.

Non-carious cervical lesions result from the loss of tooth structure in the cervical region of the vestibular surface of the dental elements, not related to caries. With the objective to verify the clinical and epidemiological features and risk factors of these injuries were evaluated 300 dental elements on 96 adult patients that were subjected to interview and intraoral clinical examination. The results presented a prevalence ratio in which the 0,87 of patients evaluated are prosthetic users. In oral hygiene habits, 0,51 use soft brush, 0,52 brush three times a day and 0,68 use brushing direction combined. Was evaluated in these patients that 0,56 have a history of gastrointestinal pathologies; 40% made use of medications and 0,36 have disocclusion characteristics in canine guide. In relation to the characteristics of the lesion, 0,40 have type of contact, 43% with a depth less than 1 mm and 0,13 are retainers of removable partial denture. As for the eating habits, 0,93 con-sume citrus fruits and 0,15 are smokers. Regarding parafunctional habits, 0,35 clench your teeth; 0,36 have bruxism and 0,10 bite objects. These lesions showed a higher prevalence in premolars 0,54.Verified the clinical and epidemiological features and risk factors of non-carious cervical lesions, we conclude that the determining factor for the emergence of these lesions has shown characteristics multifactorial, with highest percentage for patients with occlusal instability, intake of acidic drinks, prosthesis, gastrointestinal diseases and parafunctional habits.

As lesões cervicais não cariosas residem da perda de estrutura dentária na região cervical da superfície vestibular dos elementos dentários, não relacionados com a lesão de cárie. Objetivo: verificar as características clinicas e epidemiológi-cas, e os fatores de risco destas lesões. Materiais e métodos: foram avaliados 300 elementos dentários em 96 indivíduos adultos que foram submetidos a entrevista e exame clinico intra-oral. Resultados: se encontrou uma razão de prevalência em que 0,87 dos pacientes avaliados são usuários de prótese. Em hábitos de higiene oral, 0,51 utilizam escova macia, 0,52 escovam três vezes ao dia e 0,68 utilizam direção de escovação combinada. Avaliou-se nos pacientes que, 0,56 apresentam histórico de patologias gastrointestinais; 40% faziam uso de medicamentos e 0,36 apresentam características de desoclusão em guia canino. Com relação as características da lesão, 0,40 apresentam tipo de contato A, 43% com profundidade menor que 1mm e 0,13 são retentores de prótese parcial removível. Quanto aos hábitos alimentares, 0,93 consomem bebidas cítricas e 0,15 são fumantes. Referente aos hábitos parafuncionais, 0,35 apertam os dentes; 0,36 possuem bruxismo e 0,10 mordem objetos. Estas lesões apresentaram uma maior prevalência em pré-molares 0,54. Verifi-cado as características clinicas e epidemiológicas, e os fatores de risco das lesões cervicais não cariosas, conclui-se que o fator determinante para o surgimento destas lesões demonstrou características multifatoriais, com maior percentual para pacientes com instabilidade oclusal dental, ingestão de bebidas ácidas, usuários de prótese, patologias gastrointestinais e hábitos parafuncionais.

The acetate uptake transporter family motif "NPAPLGL(M/S)" is essential for substrate uptake.

Organic acids are recognized as one of the most prevalent compounds in ecosystems, thus the transport and assimilation of these molecules represent an adaptive advantage for organisms. The AceTr family members are associated with the active transport of organic acids, namely acetate and succinate. The phylogenetic analysis shows this family is dispersed in the tree of life. However, in eukaryotes, it is almost limited to microbes, though reaching a prevalence close to 100% in fungi, with an essential role in spore development. Aiming at deepening the knowledge in this family, we studied the acetate permease AceP from Methanosarcina acetivorans, as the first functionally characterized archaeal member of this family. Furthermore, we demonstrate that the yeast Gpr1 from Yarrowia lipolytica is an acetate permease, whereas the Ady2 closest homologue in Saccharomyces cerevisiae, Fun34, has no role in acetate uptake. In this work, we describe the functional role of the AceTr conserved motif NPAPLGL(M/S). We further unveiled the role of the amino acid residues R122 and Q125 of SatP as essential for protein activity.

Pseudomonas fluorescens tolerance to benzyldimethyldodecyl ammonium chloride: Altered phenotype and cross-resistance.

OBJECTIVES: Benzyldimethyldodecyl ammonium chloride (BDMDAC) is a quaternary ammonium compound (QAC) with bactericidal action that is used as an active molecule in detergent formulations. Pseudomonas fluorescens is a Gram-negative bacterium with versatile metabolism that is frequently present in biofilms on industrial surfaces. This work reports P. fluorescens adaptation to BDMDAC and subsequent concurrent reduced susceptibility to the QAC benzalkonium chloride (BAC) and the antimicrobial ciprofloxacin (CIP). METHODS: Stepwise adaptation to increasing concentrations of BDMDAC was easily achieved and caused changes in the bacterial phenotype of P. fluorescens. Adaptation was evaluated through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination and was subsequently confirmed by time-kill curves. Biofilm phenotype (biomass and number of cells) was characterised for the adapted and reference strains after treatment with BDMDAC, BAC and CIP. RESULTS: Susceptibility to BAC and CIP was reduced in adapted P. fluorescens. Biofilms developed by the adapted strain had 20% more mass and a higher number of bacteria (2 log). CONCLUSIONS: This study revealed that exposure to sublethal concentrations of BDMDAC may select tolerant strains to that product as well as to related products and unrelated antimicrobial agents.

Feed restriction and a diet's caloric value: The influence on the aerobic and anaerobic capacity of rats.

BACKGROUND: The influence of feed restriction and different diet's caloric value on the aerobic and anaerobic capacity is unclear in the literature. Thus, the objectives of this study were to determine the possible influences of two diets with different caloric values and the influence of feed restriction on the aerobic (anaerobic threshold: AT) and anaerobic (time to exhaustion: Tlim) variables measured by a lactate minimum test (LM) in rats. METHODS: We used 40 adult Wistar rats. The animals were divided into four groups: ad libitum commercial Purina® diet (3028.0 Kcal/kg) (ALP), restricted commercial Purina® diet (RAP), ad libitum semi-purified AIN-93 diet (3802.7 Kcal/kg) (ALD) and restricted semi-purified AIN-93 diet (RAD). The animals performed LM at the end of the experiment, 48 h before euthanasia. Comparisons between groups were performed by analysis of variance (p < 0,05). RESULTS: At the end of the experiment, the weights of the rats in the groups with the restricted diets were significantly lower than those in the groups with ad libitum diet intakes. In addition, the ALD group had higher amounts of adipose tissue. With respect to energetic substrates, the groups subjected to diet restriction had significantly higher levels of liver and muscle glycogen. There were no differences between the groups with respect to AT; however, the ALD group had lower lactatemia at the AT intensity and higher Tlim than the other groups. CONCLUSIONS: We conclude that dietary restriction induces changes in energetic substrates and that ad libitum intake of a semi-purified AIN-93 diet results in an increase in adipose tissue, likely reducing the density of the animals in water and favouring their performance during the swimming exercises.