Recent trends in molecular diagnostics of yeast infections : from PCR to NGS

The incidence of opportunistic yeast infections in humans has been increasing over recent years. These infections are difficult to treat and diagnose, in part due to the large number and broad diversity of species that can underlie the infection. In addition, resistance to one or several antifungal drugs in infecting strains is increasingly being reported, severely limiting therapeutic options and showcasing the need for rapid detection of the infecting agent and its drug susceptibility profile. Current methods for species and resistance identification lack satisfactory sensitivity and specificity, and often require prior culturing of the infecting agent, which delays diagnosis. Recently developed high-throughput technologies such as next generation sequencing or proteomics are opening completely new avenues for more sensitive, accurate and fast diagnosis of yeast pathogens. These approaches are the focus of intensive research, but translation into the clinics requires overcoming important challenges. In this review, we provide an overview of existing and recently emerged approaches that can be used in the identification of yeast pathogens and their drug resistance profiles. Throughout the text we highlight the advantages and disadvantages of each methodology and discuss the most promising developments in their path from bench to bedside

Biosynthesis of Vitamin C by Yeast Leads to Increased Stress Resistance

during respiration, or indirectly-caused by other stressing factors. Vitamin C or L-ascorbic acid acts as a scavenger of ROS, thereby potentially protecting cells from harmful oxidative products. While most eukaryotes synthesize ascorbic acid, yeast cells produce erythro-ascorbic acid instead. The actual importance of this antioxidant substance for the yeast is still a subject of scientific debate. is increased, but also the tolerance to low pH and weak organic acids at low pH is increased. cells endogenously producing vitamin C as a cellular model to study the genesis/protection of ROS as well as genotoxicity

Prevalence of fibromyalgia in a low socioeconomic status population

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to estimate the prevalence of fibromyalgia, as well as to assess the major symptoms of this syndrome in an adult, low socioeconomic status population assisted by the primary health care system in a city in Brazil.</p> <p>Methods</p> <p>We cross-sectionally sampled individuals assisted by the public primary health care system (n = 768, 35–60 years old). Participants were interviewed by phone and screened about pain. They were then invited to be clinically assessed (304 accepted). Pain was estimated using a Visual Analogue Scale (VAS). Fibromyalgia was assessed using the Fibromyalgia Impact Questionnaire (FIQ), as well as screening for tender points using dolorimetry. Statistical analyses included Bayesian Statistics and the Kruskal-Wallis Anova test (significance level = 5%).</p> <p>Results</p> <p>From the phone-interview screening, we divided participants (n = 768) in three groups: No Pain (NP) (n = 185); Regional Pain (RP) (n = 388) and Widespread Pain (WP) (n = 106). Among those participating in the clinical assessments, (304 subjects), the prevalence of fibromyalgia was 4.4% (95% confidence interval [2.6%; 6.3%]). Symptoms of pain (VAS and FIQ), feeling well, job ability, fatigue, morning tiredness, stiffness, anxiety and depression were statically different among the groups. In multivariate analyses we found that individuals with FM and WP had significantly higher impairment than those with RP and NP. FM and WP were similarly disabling. Similarly, RP was no significantly different than NP.</p> <p>Conclusion</p> <p>Fibromyalgia is prevalent in the low socioeconomic status population assisted by the public primary health care system. Prevalence was similar to other studies (4.4%) in a more diverse socioeconomic population. Individuals with FM and WP have significant impact in their well being.</p

Monitoring Procalcitonin in Febrile Neutropenia: What Is Its Utility for Initial Diagnosis of Infection and Reassessment in Persistent Fever?

Background: Management of febrile neutropenic episodes (FE) is challenged by lacking microbiological and clinical documentation of infection. We aimed at evaluating the utility of monitoring blood procalcitonin (PCT) in FE for initial diagnosis of infection and reassessment in persistent fever.Methods: PCT kinetics was prospectively monitored in 194 consecutive FE (1771 blood samples): 65 microbiologically documented infections (MDI, 33.5%; 49 due to non-coagulase-negative staphylococci, non-CNS), 68 clinically documented infections (CDI, 35%; 39 deep-seated), and 61 fever of unexplained origin (FUO, 31.5%).Results: At fever onset median PCT was 190 pg/mL (range 30-26'800), without significant difference among MDI, CDI and FUO. PCT peak occurred on day 2 after onset of fever: non-CNS-MDI/deep-seated-CDI (656, 80-86350) vs. FUO (205, 33-771; p&lt;0.001). PCT &gt;500 pg/mL distinguished non-CNS-MDI/deep-seated-CDI from FUO with 56% sensitivity and 90% specificity. PCT was &gt;500 pg/ml in only 10% of FUO (688, 570-771). A PCT peak &gt;500 pg/mL (1196, 524-11950) occurred beyond 3 days of persistent fever in 17/21 (81%) invasive fungal diseases (IFD). This late PCT peak identified IFD with 81% sensitivity and 57% specificity and preceded diagnosis according to EORTC-MSG criteria in 41% of cases. In IFD responding to therapy, median days to PCT &lt;500 pg/mL and defervescence were 5 (1-23) vs. 10 (3-22; p = 0.026), respectively.Conclusion: While procalcitonin is not useful for diagnosis of infection at onset of neutropenic fever, it may help to distinguish a minority of potentially severe infections among FUOs on day 2 after onset of fever. In persistent fever monitoring procalcitonin contributes to early diagnosis and follow-up of invasive mycose

Sprouted Innervation into Uterine Transplants Contributes to the Development of Hyperalgesia in a Rat Model of Endometriosis

Endometriosis is an enigmatic painful disorder whose pain symptoms remain difficult to alleviate in large part because the disorder is defined by extrauteral endometrial growths whose contribution to pain is poorly understood. A rat model (ENDO) involves autotransplanting on abdominal arteries uterine segments that grow into vascularized cysts that become innervated with sensory and sympathetic fibers. ENDO rats exhibit vaginal hyperalgesia. We used behavioral, physiological, and immunohistochemical methods to test the hypothesis that cyst innervation contributes to the development of this hyperalgesia after transplant. Rudimentary sensory and sympathetic innervation appeared in the cysts at two weeks, sprouted further and more densely into the cyst wall by four weeks, and matured by six weeks post-transplant. Sensory fibers became abnormally functionally active between two and three weeks post-transplant, remaining active thereafter. Vaginal hyperalgesia became significant between four and five weeks post-transplant, and stabilized after six to eight weeks. Removing cysts before they acquired functional innervation prevented vaginal hyperalgesia from developing, whereas sham cyst removal did not. Thus, abnormally-active innervation of ectopic growths occurs before hyperalgesia develops, supporting the hypothesis. These findings suggest that painful endometriosis can be classified as a mixed inflammatory/neuropathic pain condition, which opens new avenues for pain relief. The findings also have implications beyond endometriosis by suggesting that functionality of any transplanted tissue can be influenced by the innervation it acquires

Dietary supplementation with hydrolyzed yeast and its effect on the performance, intestinal microbiota, and immune response of weaned piglets.

The objective of this study was to evaluate the effects of autolyzed yeast on performance, cecal microbiota, and leukogram of weaned piglets. A total of 96 piglets of commercial line weaned at 21-day-old were used. The experimental design was a randomized block design with four treatments (diets containing 0.0%, 0.3%, 0.6%, and 0.9% autolyzed yeast), eight replicates, and three animals per pen in order to evaluate daily weight gain, daily feed intake, and feed conversion in periods of 0 to 15, 0 to 26, and 0 to 36 days. Quadratic effects of autolyzed yeast inclusion were observed on the feed conversion from 0 to 15 days, on daily weight gain from 0 to 15 days, 0 to 26 days and, 0 to 36 days, indicating an autolyzed yeast optimal inclusion level between 0.4% and 0.5%. No effect from autolyzed yeast addition was observed on piglet daily feed intake, cecal microbiota, and leukogram; however, i.m. application of E. coli lipopolysaccharide reduced the values of total leukocytes and their fractions (neutrophils, eosinophils, lymphocytes, monocytes, and rods). Therefore, autolyzed yeast when provided at levels between 0.4% and 0.5% improved weaned piglets’ performance.info:eu-repo/semantics/publishedVersio

A Mitosis Block Links Active Cell Cycle with Human Epidermal Differentiation and Results in Endoreplication

How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines

Conditional Transgenesis Using Dimerizable Cre (DiCre)

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCre×R26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems