Czech Academy of Sciences, Institute of Biotechnology

BACKGROUND: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. CONTENT: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. SUMMARY: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.

A GENTSthatcanextendthe lifespanofmiceareof interestfortworeasons:theycanprovidenewmodels of delayed aging to teach us more about what controls agingrateandhowagingleadstodisease;andinaddition, they serve as a first step toward eventual development of pharmaceuticals to slow aging and retard diseases in humans. The National Institute on Aging Intervention Testingprogram(ITP)haspreviouslyreportedsignificant increases in life span caused by aspirin and nordihydroguaiareticacid inmalemice(1)andbyrapamycinin bothmaleandfemalemice(2).ThedesignoftheITP(3) emphasizestheuseofgeneticallyheterogeneousmiceto mitigate against idiosyncrasies that can complicate inter-pretationofdatafromasingleinbredorF1hybridstock andincludesparallelreplicationofprotocolsatthreesites, the University of Texas (UT), University of Michigan (UM), and The Jackson Laboratory (TJL), with standard operatingprotocolsthatattempttoreproducekeyelements of the environmental conditions at each site. Sufficient numbers of mice are used in each yearly cohort to give morethan80%powertodetectanincreaseordecreaseof 10%inmeanlifespan,withrespecttocontrolsofthesame sex,evenifonlytwoofthethreesitescancontributedata tothepooledanalysis.

Polar flow of the phytohormone auxin requires plasma membrane-associated PIN proteins and underlies multiple developmental processes in plants. Here we address the importance of the polarity of subcellular PIN localization for the directionality of auxin transport in Arabidopsis thaliana. Expression of different PINs in the root epidermis revealed the importance of PIN polar positions for directional auxin flow and root gravitropic growth. Interfering with sequence-embedded polarity signals directly demonstrates that PIN polarity is a primary factor in determining the direction of auxin flow in meristematic tissues. This finding provides a crucial piece in the puzzle of how auxin flow can be redirected via rapid changes in PIN polarity.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

Systemic absorption and metabolism of drugs in the small intestine, metabolism by the liver as well as excretion by the kidney are key determinants of efficacy and safety for therapeutic candidates. However, these systemic responses of applied substances lack in most in vitro assays. In this study, a microphysiological system maintaining the functionality of four organs over 28 days in co-culture has been established at a minute but standardized microsystem scale. Preformed human intestine and skin models have been integrated into the four-organ-chip on standard cell culture inserts at a size 100,000-fold smaller than their human counterpart organs. A 3D-based spheroid, equivalent to ten liver lobules, mimics liver function. Finally, a barrier segregating the media flow through the organs from fluids excreted by the kidney has been generated by a polymeric membrane covered by a monolayer of human proximal tubule epithelial cells. A peristaltic on-chip micropump ensures pulsatile media flow interconnecting the four tissue culture compartments through microfluidic channels. A second microfluidic circuit ensures drainage of the fluid excreted through the kidney epithelial cell layer. This four-organ-chip system assures near to physiological fluid-to-tissue ratios. In-depth metabolic and gene analysis revealed the establishment of reproducible homeostasis among the co-cultures within two to four days, sustainable over at least 28 days independent of the individual human cell line or tissue donor background used for each organ equivalent. Lastly, 3D imaging two-photon microscopy visualised details of spatiotemporal segregation of the two microfluidic flows by proximal tubule epithelia. To our knowledge, this study is the first approach to establish a system for in vitro microfluidic ADME profiling and repeated dose systemic toxicity testing of drug candidates over 28 days.

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.

BACKGROUND: The association of body-mass index (BMI) from adolescence to adulthood with obesity-related diseases in young adults has not been completely delineated. METHODS: We conducted a prospective study in which we followed 37,674 apparently healthy young men for incident angiography-proven coronary heart disease and diabetes through the Staff Periodic Examination Center of the Israeli Army Medical Corps. The height and weight of participants were measured at regular intervals, with the first measurements taken when they were 17 years of age. RESULTS: During approximately 650,000 person-years of follow-up (mean follow-up, 17.4 years), we documented 1173 incident cases of type 2 diabetes and 327 of coronary heart disease. In multivariate models adjusted for age, family history, blood pressure, lifestyle factors, and biomarkers in blood, elevated adolescent BMI (the weight in kilograms divided by the square of the height in meters; mean range for the first through last deciles, 17.3 to 27.6) was a significant predictor of both diabetes (hazard ratio for the highest vs. the lowest decile, 2.76; 95% confidence interval [CI], 2.11 to 3.58) and angiography-proven coronary heart disease (hazard ratio, 5.43; 95% CI, 2.77 to 10.62). Further adjustment for BMI at adulthood completely ablated the association of adolescent BMI with diabetes (hazard ratio, 1.01; 95% CI, 0.75 to 1.37) but not the association with coronary heart disease (hazard ratio, 6.85; 95% CI, 3.30 to 14.21). After adjustment of the BMI values as continuous variables in multivariate models, only elevated BMI in adulthood was significantly associated with diabetes (β=1.115, P=0.003; P=0.89 for interaction). In contrast, elevated BMI in both adolescence (β=1.355, P=0.004) and adulthood (β=1.207, P=0.03) were independently associated with angiography-proven coronary heart disease (P=0.048 for interaction). CONCLUSIONS: An elevated BMI in adolescence--one that is well within the range currently considered to be normal--constitutes a substantial risk factor for obesity-related disorders in midlife. Although the risk of diabetes is mainly associated with increased BMI close to the time of diagnosis, the risk of coronary heart disease is associated with an elevated BMI both in adolescence and in adulthood, supporting the hypothesis that the processes causing incident coronary heart disease, particularly atherosclerosis, are more gradual than those resulting in incident diabetes. (Funded by the Chaim Sheba Medical Center and the Israel Defense Forces Medical Corps.).

"Serum ferritin" presents a paradox, as the iron storage protein ferritin is not synthesised in serum yet is to be found there. Serum ferritin is also a well known inflammatory marker, but it is unclear whether serum ferritin reflects or causes inflammation, or whether it is involved in an inflammatory cycle. We argue here that serum ferritin arises from damaged cells, and is thus a marker of cellular damage. The protein in serum ferritin is considered benign, but it has lost (i.e. dumped) most of its normal complement of iron which when unliganded is highly toxic. The facts that serum ferritin levels can correlate with both disease and with body iron stores are thus expected on simple chemical kinetic grounds. Serum ferritin levels also correlate with other phenotypic readouts such as erythrocyte morphology. Overall, this systems approach serves to explain a number of apparent paradoxes of serum ferritin, including (i) why it correlates with biomarkers of cell damage, (ii) why it correlates with biomarkers of hydroxyl radical formation (and oxidative stress) and (iii) therefore why it correlates with the presence and/or severity of numerous diseases. This leads to suggestions for how one might exploit the corollaries of the recognition that serum ferritin levels mainly represent a consequence of cell stress and damage.

BACKGROUND: Although epidemiologic studies have suggested that several genetic variants increase the risk of myocardial infarction, large-scale association studies that examine many polymorphisms simultaneously are required to allow reliable prediction of the genetic risk of myocardial infarction. METHODS: We used a fluorescence- or colorimetry-based allele-specific DNA-primer-probe assay system to determine the genotypes of 112 polymorphisms of 71 candidate genes in 2819 unrelated Japanese patients with myocardial infarction (2003 men and 816 women) and 2242 unrelated Japanese controls (1306 men and 936 women). RESULTS: In an initial screening of the 112 polymorphisms for an association with myocardial infarction in 909 subjects, 19 polymorphisms were selected in men and 18 in women by means of logistic-regression analysis, after adjustment for age, body-mass index, and the prevalence of smoking, hypertension, diabetes mellitus, hypercholesterolemia, and hyperuricemia. In a large-scale study involving the selected polymorphisms and the remaining 4152 subjects, similar logistic-regression analysis revealed that the risk of myocardial infarction was significantly associated with the C1019T polymorphism in the connexin 37 gene (P<0.001) in men and the 4G-668/5G polymorphism in the plasminogen-activator inhibitor type 1 gene (P<0.001) and the 5A-1171/6A polymorphism in the stromelysin-1 gene (P<0.001) in women. CONCLUSIONS: Determination of the genotypes of the connexin 37, plasminogen-activator inhibitor type 1, and stromelysin-1 genes may prove reliable in predicting the genetic risk of myocardial infarction and might thus contribute to the primary prevention of this condition.

MicroRNAs (miRNAs) are endogenously expressed RNAs, 18-25 nucleotides in length, that repress protein translation through binding to target mRNAs. miRNAs have been implicated in many cellular processes including cell proliferation, differentiation, and death. Recently, let-7 miRNAs were found to regulate human RAS oncogene expression and to be often down-regulated in human lung tumors. In this study, we examined the expression of let-7 miRNAs in human colon cancer tumors and cell lines, with the result that 2 of 6 cases and 1 of 3 cell lines showed reduced expression of let-7. When let-7 low-expressing DLD-1 human colon cancer cells were transfected with let-7a-1 precursor miRNA, which is located at chromosome 9q22.3, the cells underwent significant growth suppression. At that time, the levels of RAS and c-myc proteins were lowered after the transfection, whereas the levels of both of their mRNAs remained almost unchanged. These findings suggest the involvement of let-7 miRNA in the growth of colon cancer cells. Thus, miRNAs might provide a basis for novel RNA anti-cancer agents.

We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range.

Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

This review summarizes recent advances in our knowledge of the glomerular filter and the causes of hereditary proteinuria syndromes.

To construct an East Asia mitochondrial DNA (mtDNA) phylogeny, we sequenced the complete mitochondrial genomes of 672 Japanese individuals (http://www.giib.or.jp/mtsnp/index_e.html). This allowed us to perform a phylogenetic analysis with a pool of 942 Asiatic sequences. New clades and subclades emerged from the Japanese data. On the basis of this unequivocal phylogeny, we classified 4713 Asian partial mitochondrial sequences, with <10% ambiguity. Applying population and phylogeographic methods, we used these sequences to shed light on the controversial issue of the peopling of Japan. Population-based comparisons confirmed that present-day Japanese have their closest genetic affinity to northern Asian populations, especially to Koreans, which finding is congruent with the proposed Continental gene flow to Japan after the Yayoi period. This phylogeographic approach unraveled a high degree of differentiation in Paleolithic Japanese. Ancient southern and northern migrations were detected based on the existence of basic M and N lineages in Ryukyuans and Ainu. Direct connections with Tibet, parallel to those found for the Y-chromosome, were also apparent. Furthermore, the highest diversity found in Japan for some derived clades suggests that Japan could be included in an area of migratory expansion to Continental Asia. All the theories that have been proposed up to now to explain the peopling of Japan seem insufficient to accommodate fully this complex picture.

Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca(2+) ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.

This is a continuity of a series of taxonomic papers where materials are examined, described and novel combinations are proposed where necessary to improve our traditional species concepts and provide updates on their classification. In addition to extensive morphological descriptions and appropriate asexual and sexual connections, DNA sequence data are also analysed from concatenated datasets (rDNA, TEF-α, RBP2 and β-Tubulin) to infer phylogenetic relationships and substantiate systematic position of taxa within appropriate ranks. Wherever new species or combinations are being proposed, we apply an integrative approach (morphological and molecular data as well as ecological features wherever applicable). Notes on 125 fungal taxa are compiled in this paper, including eight new genera, 101 new species, two new combinations, one neotype, four reference specimens, new host or distribution records for eight species and one alternative morphs. The new genera introduced in this paper are Alloarthopyrenia, Arundellina, Camarosporioides, Neomassaria, Neomassarina, Neotruncatella, Paracapsulospora and Pseudophaeosphaeria. The new species are Alfaria spartii, Alloarthopyrenia italica, Anthostomella ravenna, An. thailandica, Arthrinium paraphaeospermum, Arundellina typhae, Aspergillus koreanus, Asterina cynometrae, Bertiella ellipsoidea, Blastophorum aquaticum, Cainia globosa, Camarosporioides phragmitis, Ceramothyrium menglunense, Chaetosphaeronema achilleae, Chlamydotubeufia helicospora, Ciliochorella phanericola, Clavulinopsis aurantiaca, Colletotrichum insertae, Comoclathris italica, Coronophora myricoides, Cortinarius fulvescentoideus, Co. nymphatus, Co. pseudobulliardioides, Co. tenuifulvescens, Cunninghamella gigacellularis, Cyathus pyristriatus, Cytospora cotini, Dematiopleospora alliariae, De. cirsii, Diaporthe aseana, Di. garethjonesii, Distoseptispora multiseptata, Dis. tectonae, Dis. tectonigena, Dothiora buxi, Emericellopsis persica, Gloniopsis calami, Helicoma guttulatum, Helvella floriforma, H. oblongispora, Hermatomyces subiculosa, Juncaceicola italica, Lactarius dirkii, Lentithecium unicellulare, Le. voraginesporum, Leptosphaeria cirsii, Leptosphaeria irregularis, Leptospora galii, Le. thailandica, Lindgomyces pseudomadisonensis, Lophiotrema bambusae, Lo. fallopiae, Meliola citri-maximae, Minimelanolocus submersus, Montagnula cirsii, Mortierella fluviae, Muriphaeosphaeria ambrosiae, Neodidymelliopsis ranunculi, Neomassaria fabacearum, Neomassarina thailandica, Neomicrosphaeropsis cytisi, Neo. cytisinus, Neo. minima, Neopestalotiopsis cocoës, Neopestalotiopsis musae, Neoroussoella lenispora, Neotorula submersa, Neotruncatella endophytica, Nodulosphaeria italica, Occultibambusa aquatica, Oc. chiangraiensis, Ophiocordyceps hemisphaerica, Op. lacrimoidis, Paracapsulospora metroxyli, Pestalotiopsis sequoiae, Peziza fruticosa, Pleurotrema thailandica, Poaceicola arundinis, Polyporus mangshanensis, Pseudocoleophoma typhicola, Pseudodictyosporium thailandica, Pseudophaeosphaeria rubi, Purpureocillium sodanum, Ramariopsis atlantica, Rhodocybe griseoaurantia, Rh. indica, Rh. luteobrunnea, Russula indoalba, Ru. pseudoamoenicolor, Sporidesmium aquaticivaginatum, Sp. olivaceoconidium, Sp. pyriformatum, Stagonospora forlicesenensis, Stagonosporopsis centaureae, Terriera thailandica, Tremateia arundicola, Tr. guiyangensis, Trichomerium bambusae, Tubeufia hyalospora, Tu. roseohelicospora and Wojnowicia italica. New combinations are given for Hermatomyces mirum and Pallidocercospora thailandica. A neotype is proposed for Cortinarius fulvescens. Reference specimens are given for Aquaphila albicans, Leptospora rubella, Platychora ulmi and Meliola pseudosasae, while new host or distribution records are provided for Diaporthe eres, Di. siamensis, Di. foeniculina, Dothiorella iranica, Do. sarmentorum, Do. vidmadera, Helvella tinta and Vaginatispora fuckelii, with full taxonomic details. An asexual state is also reported for the first time in Neoacanthostigma septoconstrictum. This paper contributes to a more comprehensive update and improved identification of many ascomycetes and basiodiomycetes.

BACKGROUND: Idiopathic membranous nephropathy is a major cause of the nephrotic syndrome in adults, but its etiologic basis is not fully understood. We investigated the genetic basis of biopsy-proven cases of idiopathic membranous nephropathy in a white population. METHODS: We performed independent genomewide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. Associations were calculated by means of a chi-square basic allele test; the threshold for significance was adjusted for multiple comparisons (with the Bonferroni method). RESULTS: In a joint analysis of data from the 556 patients studied (398 men), we identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A(2) receptor (PLA(2)R1) (SNP rs4664308, P=8.6×10(-29)), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0×10(-93)). The association with HLA-DQA1 was significant in all three populations (P=1.8×10(-9), P=5.6×10(-27), and P=5.2×10(-36) in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2). CONCLUSIONS: An HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of PLA2R1. Our findings suggest a basis for understanding this disease and illuminate how adaptive immunity is regulated by HLA.

Ferroptosis is a newly defined form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Erastin, the ferroptosis activator, binds to voltage-dependent anion channels VDAC2 and VDCA3, but treatment with erastin can result in the degradation of the channels. Here, the authors show that Nedd4 is induced following erastin treatment, which leads to the ubiquitination and subsequent degradation of the channels. Depletion of Nedd4 limits the protein degradation of VDAC2/3, which increases the sensitivity of cancer cells to erastin. By understanding the molecular mechanism of erastin-induced cellular resistance, we can discover how cells adapt to new molecules to maintain homeostasis. Furthermore, erastin-induced resistance mediated by FOXM1-Nedd4-VDAC2/3 negative feedback loop provides an initial framework for creating avenues to overcome the drug resistance of ferroptosis activators.

Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold, and freezing stress. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y, S, and K segments. The K segment representing a highly conserved 15 amino acid motif forming amphiphilic α-helix is especially important since it has been found in all dehydrins. Since more than 20 years, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made towards the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.