Dispersion scan frequency resolved optical gating for consistency check of pulse retrieval.

Many methods commonly used to characterize ultrafast laser pulses, such as the frequency-resolved optical gating (FROG) or the dispersion scan (d-scan), face problems when they are used on pulses with a spectrum or phase varying within the laser beam cross section or the acquisition time. The presence of such pulse shape variation leads to discrepancy between the measured FROG trace and its reconstructed counterparts. Nevertheless, it is difficult to reliably discern this shape variation because even the distorted experimental FROG trace can be reasonably reproduced by a realistic pulse shape. In this work, we examine and discern the variation of the pulse shape based on a new method, dispersion-scan FROG (D-FROG), which combines the idea of dispersion scanning with the FROG method. This technique provides a means of careful evaluation of the laser pulse based on a set of FROG traces connected by known dispersion changes. Therefore, this method can disclose seemingly correct pulse retrievals from distorted datasets. The D-FROG method can be used as a simple extension of the FROG technique to provide a consistency check able to identify the shortcomings in the pulse characterization.

Lsp1 partially substitutes for Pil1 function in eisosome assembly under stress conditions.

Eisosomes are large hemitubular structures that underlie the invaginated microdomains in the plasma membrane of various ascomycetous fungi, lichens and unicellular algae. In fungi, they are organized by BAR-domain containing proteins of the Pil1 family. Two such proteins, Pil1 and Lsp1, participate in eisosome formation in the yeast Saccharomyces cerevisiae. Under normal laboratory conditions, deletion of the PIL1 gene results in the inability of cells to assemble wild-type-like eisosomes. We found that under certain stress conditions, Lsp1 partially substitutes for the Pil1 function and mediates assembly of eisosomes, specifically following a decrease in the activity of serine palmitoyltransferase, for example, in response to hyperosmotic stress. Besides Lsp1, the assembly of eisosomes lacking Pil1 also requires Seg1 and Nce102 proteins. Using next-generation sequencing, we found that the seg1Δnce102Δpil1Δ strain, which is unable to form eisosomes, overexpresses genes coding for proteins of oxidative phosphorylation and tricarboxylic acid cycle. By contrast, genes involved in DNA repair, ribosome biogenesis and cell cycle are downregulated. Our results identify Lsp1 as a stress-responsive eisosome organizer and indicate several novel functional connections between the eisosome and essential cellular processes.

Risk factors for tumors or leukemia development in the first two years of life.

OBJECTIVES: The objective of this study was to determine the incidence of neoplastic diseases and associated risk factors in the early stages of life. METHODS: Data were retrospectively assessed in 730,000 live births between 2000 and 2019. The occurrence of tumors was monitored in the neonatal, infant (1-12 months), and toddler (13-24 months) periods. Risk factors were divided into demographic, internal, and environmental factors. The control group consisted of subjects in the same age category without oncological diseases. RESULTS: A total of 452 neoplastic diseases were diagnosed in the study sample. In total, 24% (110/452) manifested during the neonatal period, 45% (203/452) in infants, and 31% (139/452) at the age of 13-24 months. Any genetic disease (OR 26.68; 95% CI 7.64-93.12) and medications used by the mother (OR 3.07; 95% CI 1.32-7.15) were identified as risk factors. Without adjustment for all factors, asphyxia in the first minute, a younger age of the mother, lower pregnancy, and the presence of a congenital defect manifested themselves as risk factors. CONCLUSIONS: The highest risk factors for the development of early childhood tumors were identified as with medications used by the mother before or during pregnancy and genetic diseases.

Two Different Phospholipases C, Isc1 and Pgc1, Cooperate To Regulate Mitochondrial Function.

The absence of Isc1, the yeast homologue of mammalian neutral sphingomyelinase type 2, leads to severe mitochondrial dysfunction. We show that the deletion of another type C phospholipase, the phosphatidylglycerol (PG)-specific phospholipase Pgc1, rescues this defect. Phosphatidylethanolamine (PE) levels and cytochrome c oxidase activity, which were reduced in isc1Δ cells, were restored to wild-type levels in the pgc1Δ isc1Δ mutant. The Pgc1 substrate PG inhibited the in vitro activities of Isc1 and the phosphatidylserine decarboxylase Psd1, an enzyme crucial for PE biosynthesis. We also identify a mechanism by which the balance between the current demand for PG and its consumption is controlled. We document that the product of PG hydrolysis, diacylglycerol, competes with the substrate of PG-phosphate synthase, Pgs1, and thereby inhibits the biosynthesis of excess PG. This feedback loop does not work in the absence of Pgc1, which catalyzes PG degradation. Finally, Pgc1 activity is partially inhibited by products of Isc1-mediated hydrolysis. The described functional interconnection of the two phospholipases contributes significantly to lipid homeostasis throughout the cellular architecture. IMPORTANCE In eukaryotic cells, mitochondria are constantly adapting to changes in the biological activity of the cell, i.e., changes in nutrient availability and environmental stresses. We propose a model in which this adaptation is mediated by lipids. Specifically, we show that mitochondrial phospholipids regulate the biosynthesis of cellular sphingolipids and vice versa. To do this, lipids move by free diffusion, which does not require energy and works under any condition. This model represents a simple way for the cell to coordinate mitochondrial structure and performance with the actual needs of overall cellular metabolism. Its simplicity makes it a universally applicable principle of cellular regulation.

Microdomain Protein Nce102 Is a Local Sensor of Plasma Membrane Sphingolipid Balance.

Sphingolipids are essential building blocks of eukaryotic membranes and important signaling molecules that are regulated tightly in response to environmental and physiological inputs. While their biosynthetic pathway has been well-described, the mechanisms that facilitate the perception of sphingolipid levels at the plasma membrane remain to be uncovered. In Saccharomyces cerevisiae, the Nce102 protein has been proposed to function as a sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that the production of Nce102 increases following sphingolipid biosynthesis inhibition and that Nce102 is internalized when excess sphingolipid precursors are supplied. This finding suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. The physiological role of Nce102 in the regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of hydroxylated complex sphingolipids in response to heat stress in the nce102Δ deletion mutant. We also demonstrate that Nce102 behaves analogously in the widespread human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species. IMPORTANCE Microorganisms are challenged constantly by their rapidly changing environment. To survive, they have developed diverse mechanisms to quickly perceive stressful situations and adapt to them appropriately. The primary site of both stress sensing and adaptation is the plasma membrane. We identified the yeast protein Nce102 as a marker of local sphingolipid levels and fluidity in the plasma membrane. Nce102 is an important structural and functional component of the membrane compartment Can1 (MCC), a plasma membrane microdomain stabilized by a large cytosolic hemitubular protein scaffold, the eisosome. The MCC/eisosomes are widely conserved among fungi and unicellular algae. To determine if Nce102 carries out similar functions in other organisms, we analyzed the human fungal pathogen Candida albicans and found that Nce102 responds to sphingolipid levels also in this organism, which has potential applications for the development of novel therapeutic approaches. The presented study represents a valuable model for how organisms regulate plasma membrane sphingolipids.

Targeted generation of complex temporal pulse profiles.

A targeted shaping of complex femtosecond pulse waveforms and their characterization is essential for many spectroscopic applications. A 4f pulse shaper combined with an advanced pulse characterization technique should, in the idealized case, serve this purpose for an arbitrary pulse shape. This is, however, violated in the real experiment by many imperfections and limitations. Although the complex waveform generation has been studied in-depth, the comparison of the effects of various experimental factors on the actual pulse shape has stayed out of focus so far. In this paper, we present an experimental study on the targeted generation and retrieval of complex pulses by using two commonly-used techniques: spatial-light-modulator (SLM)-based 4f pulse shaper and second-harmonic generation frequency-resolved optical gating (FROG) and cross-correlation FROG (XFROG). By combining FROG and XFROG traces, we analyze the pulses with SLM-adjusted complex random phases ranging from simple to very complex waveforms. We demonstrate that the combination of FROG and XFROG ensures highly consistent pulse retrieval, irrespective of the used retrieval algorithm. This enabled us to evaluate the role of various experimental factors on the agreement between the simulated and actual pulse shape. The factors included the SLM pixelation, SLM pixel crosstalk, finite laser focal spot in the pulse shaper, or interference fringes induced by the SLM. In particular, we observe that including the SLM pixelation and crosstalk effect significantly improved the pulse shaping simulation. We demonstrate that the complete simulation can faithfully reproduce the pulse shape. Nevertheless, even in this case, the intensity of individual peaks differs between the retrieved and simulated pulses, typically by 10-20% of the peak value, with the mean standard deviation of 5-9% of the maximum pulse intensity. We discuss the potential sources of remaining discrepancies between the theoretically expected and experimentally retrieved pulse.

Blocking phosphatidylglycerol degradation in yeast defective in cardiolipin remodeling results in a new model of the Barth syndrome cellular phenotype.

Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.

Novel mutations in TRPM6 gene associated with primary hypomagnesemia with secondary hypocalcemia. Case report.

BACKGROUND: Primary hypomagnesemia with secondary hypocalcemia (HSH) is a rare genetic disorder. Dysfunctional transient receptor potential melastatin 6 causes impaired intestinal absorption of magnesium, leading to low serum levels accompanied by hypocalcemia. Typical signs at initial manifestation are generalized seizures, tetany, and/or muscle spasms. CASE REPORT: We present a 5 w/o female manifesting tonic-clonic seizures. Laboratory tests detected severe hypomagnesemia and hypocalcemia. The molecular genetic analysis revealed two novel mutations within the TRPM6 gene c.3308dupC (p.Pro1104Thrfs*28) (p.P1104Tfs*28) and c.3958C>T (p.Gln1302*) (p.Q1302*) and the patient was successfully treated with Mg supplementation. CONCLUSION: Ion disbalance should be taken into account in the differential diagnosis of infantile seizures. Accurate diagnosis of HSH together with appropriate treatment are crucial to prevent irreversible neurological outcomes.

Management After Windstorm Affects the Composition of Ectomycorrhizal Symbionts of Regenerating Trees but Not Their Mycorrhizal Networks.

Due to ongoing climate change, forests are expected to face significant disturbances more frequently than in the past. Appropriate management is intended to facilitate forest regeneration. Because European temperate forests mostly consist of trees associated with ectomycorrhizal (ECM) fungi, understanding their role in these disturbances is important to develop strategies to minimize their consequences and effectively restore forests. Our aim was to determine how traditional (EXT) and nonintervention (NEX) management in originally Norway spruce (Picea abies) forests with an admixture of European larch (Larix decidua) affect ECM fungal communities and the potential to interconnect different tree species via ECM networks 15 years after a windstorm. Ten plots in NEX and 10 plots in EXT with the co-occurrences of Norway spruce, European larch, and silver birch (Betula pendula) were selected, and a total of 57 ECM taxa were identified using ITS sequencing from ECM root tips. In both treatments, five ECM species associated with all the studied tree species dominated, with a total abundance of approximately 50% in the examined root samples. Because there were no significant differences between treatments in the number of ECM species associated with different tree species combinations in individual plots, we concluded that the management type did not have a significant effect on networking. However, management significantly affected the compositions of ECM symbionts of Norway spruce and European larch but not those of silver birch. Although this result is explained by the occurrence of seedlings and ECM propagules that were present in the original forest, the consequences are difficult to assess without knowledge of the ecology of different ECM symbionts.

Influence of the delay line jitter on the SHG FROG reconstruction.

Frequency-resolved optical gating (FROG) counts among the most used methods to characterize complex femtosecond pulses. The multishot FROG experiment, studied in this work, relies on varying a delay between two replicas of the measured pulse, where the delay accuracy can suffer from delay line imperfections, setup instability, or minimization of the acquisition time. We present a detailed study on the effect of the delay line jitter on the pulse retrieval. We carried out simulations with the jitter values ranging from high-precision delay lines (100 nm) up to extremely unstable measurements (>1000 nm). For three sets of pulses, we quantified criteria assuring reliable reconstruction, using ptychographic algorithm, of a complex pulse based on the experimentally available FROG trace error. We observe that the effect of the jitter scales together with the spectral bandwidth. However, the pulse reconstruction is relatively robust against the jitter and, even for a severe distortion of the FROG trace (e.g., a jitter of 500 nm for broadband pulses), the main features of all pulses are retrieved with high fidelity. Our results provide guidance for the limitations based on the delay imperfections in the FROG experiment.

The development and significance of microRNA sequence variants in carcinogenesis.

BACKGROUND: MicroRNA (miRNA) are a class of short non-coding RNAs that regulate gene expression at the posttranscriptional level. They are involved in key cellular processes and development as well as progression of many diseases. Their levels reflect the physiological state of organisms; therefore, the expression profiles of these molecules are analyzed in bio-marker studies. Due to their properties, miRNA appear to be promising dia-gnostic, prognostic and predictive bio-markers of cancer. Recent studies indicate the existence of sequence variants in miRNA, so-called isomiRs, which differ from the annotated miRNAs by altered sequences due to posttranscriptional modifications. These isomiRs may have a higher abundance than canonical miRNA. The characterization of isomiRs reveals their regulated distribution and different bio-logical properties and thus suggest the possible bio-logical significance of the modifications. The presence of isomiRs can also significantly affect the results of bio-marker studies. Currently, the research is focused on their possible clinical significance. PURPOSE: The aim of this review is to provide an overview of current knowledge about sequence variants in miRNA. The review summarizes the mechanisms of isomiRs bio-genesis and describes the effects of sequence heterogeneity on miRNA stability, function and analysis. Subsequently, the role of isomiRs in bio-marker studies is discussed.

Thermal Ablation and Transarterial Chemoembolization are Characterized by Changing Dynamics of Circulating MicroRNAs.

PURPOSE: To determine whether the levels of circulating microRNAs (miRNAs) are altered in patients undergoing thermal ablation and chemoembolization and whether these changes are predictive of a clinical outcome. MATERIAL AND METHODS: This prospective study consisted of 43 patients diagnosed with hepatocellular carcinoma (n = 15) and intrahepatic colorectal cancer metastases (n = 28) treated with thermal ablation (n = 23; radiofrequency [n = 6] or microwave [n = 19]), chemoembolization using drug-eluting embolics (n = 18), or both (n = 2). Four blood samples (immediately before the intervention and 60-90 minutes, 24 hours, and 7 days after the intervention) were taken to measure the plasma concentrations of miRNAs related to hypoxia (miR-21 and miR-210), liver injury (miR-122), epithelial-mesenchymal transition (miR-200a), and apoptosis (miR-34a) using miRNA-specific TaqMan assays and quantitative real-time polymerase chain reaction. Tumor burden and treatment response at 3 months were evaluated using the modified response evaluation criteria in solid tumors. The miRNA results were compared with clinical outcomes (Mann-Whitney U test, Wilcoxon matched-pair test). RESULTS: Dynamic changes in the circulating miRNA levels were observed following both the interventions. For thermal ablation, significant increases in miR-21, miR-210, miR-122, miR-200a, and miR-34a concentrations peaked 60-90 minutes after the intervention (P < .01). However, for transarterial chemoembolization, maximum increases in the miRNA concentrations were observed at 24 hours after the intervention for miR-21, miR-210, miR-122, miR-200a, and miR-34a (P < .05). The increased concentrations of the circulating miRNAs were followed by a subsequent decline to baseline by 7 days. For the thermal ablation (but not chemoembolization) patients, elevations in the miR-210 and miR-200a levels were associated with early progressive disease at 3 months (P = .040 and P = .012, respectively). CONCLUSIONS: Increased but dynamic levels of circulating miRNAs are present following interventional oncologic procedures and may prove useful as biomarkers for the monitoring of clinical outcomes.

Novel Splicing Variant in the PMM2 Gene in a Patient With PMM2-CDG Syndrome Presenting With Pericardial Effusion: A Case Report.

Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases with the phosphomannomutase 2 (PMM2)-CDG being the most common form of CDG. Most of these monogenic diseases are autosomal recessive and have multi-systemic manifestations, mainly psychomotor retardation, facial dysmorphisms, characteristic distribution of the fat pads, and variable coagulation abnormalities. The association of fetal hydrops with CDG has been reported, and pericardial effusion was also rarely observed in patients with PMM2-CDG. Here we describe an infant boy with PMM2-CDG. The diagnosis was suspected based on inverted nipples, fat pads, and combined coagulopathy. However, the primary symptom was progressive pericardial effusion leading to patient death at the age of 3 months. Screening for CDG performed by the use of isoelectric focusing of serum transferrin showed a typical PMM2-CDG pattern. Exome sequencing revealed one common pathogenic variant (c.691G > A/p.Val231Met) and one novel variant (c.447 + 3dupA) in the PMM2 gene. Both PMM2 variants were further confirmed by Sanger sequencing in both the proband and the parents' DNA. The novel variant was predicted to result in loss of donor splice site, and the analysis at mRNA level confirmed that it leads to exon five skipping (r.348_447del) and causes premature termination of translation to the protein (p.G117Kfs∗4), therefore is classified as likely pathogenic. Although there is no curative therapy for the PMM2-CDG at the moment, the other supportive care options are available to be offered. The definite diagnosis of PMM2-CDG can also assist in the process of genetic counseling, family planning, and preimplantation genetic diagnosis.

Plasma Membrane Protein Nce102 Modulates Morphology and Function of the Yeast Vacuole.

Membrane proteins are targeted not only to specific membranes in the cell architecture, but also to distinct lateral microdomains within individual membranes to properly execute their biological functions. Yeast tetraspan protein Nce102 has been shown to migrate between such microdomains within the plasma membrane in response to an acute drop in sphingolipid levels. Combining microscopy and biochemistry methods, we show that upon gradual ageing of a yeast culture, when sphingolipid demand increases, Nce102 migrates from the plasma membrane to the vacuole. Instead of being targeted for degradation it localizes to V-ATPase-poor, i.e., ergosterol-enriched, domains of the vacuolar membrane, analogous to its plasma membrane localization. We discovered that, together with its homologue Fhn1, Nce102 modulates vacuolar morphology, dynamics, and physiology. Specifically, the fusing of vacuoles, accompanying a switch of fermenting yeast culture to respiration, is retarded in the strain missing both proteins. Furthermore, the absence of either causes an enlargement of ergosterol-rich vacuolar membrane domains, while the vacuoles themselves become smaller. Our results clearly show decreased stability of the V-ATPase in the absence of either Nce102 or Fhn1, a possible result of the disruption of normal microdomain morphology of the vacuolar membrane. Therefore, the functionality of the vacuole as a whole might be compromised in these cells.

The lipid droplet protein Pgc1 controls the subcellular distribution of phosphatidylglycerol.

The biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here, we present evidence that the subcellular distribution of PG is maintained by the locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. A fluorescently labeled Pgc1 protein accumulates on the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but do not even host any phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to the membranes of the endoplasmic reticulum, mitochondria and even of artificial phospholipid vesicles. Fluorescence recovery after photobleaching analysis confirms the continuous exchange of GFP-Pgc1 within the individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate the immediate phospholipase activity of Pgc1. In this model, LD serve as a storage place and shelter Pgc1, preventing its untimely degradation, while both phospholipase activity and degradation of the enzyme occur in the membranes.

Tumor expression of miR-34a-3p is an independent predictor of recurrence in non-muscle-invasive bladder cancer and promising additional factor to improve predictive value of EORTC nomogram.

OBJECTIVES: Non-muscle-invasive bladder cancer (NMIBC) is a heterogeneous disease characterized by a high primary tumor recurrence rate. Current prognostic systems used for predicting recurrence in individual patients have limitations and do not consider the biological background of this tumor type. Our study aimed to find microRNAs (miRNAs) associated with NMIBC recurrence. METHODS: Seventy-eight NMIBC patients were prospectively enrolled and divided into exploratory and validation cohorts. Out of these patients, 32 developed recurrence within 18 months after surgery, while 46 did not show any sign of recurrence after 30 months. Expression profiles of 2,578 miRNAs were obtained using Affymetrix miRNA microarrays and candidate miRNAs validated using the individual quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: The expression profiling revealed a set of 137 miRNAs differentially expressed between NMIBC patients with and without recurrence (P < 0.05). In the validation phase, miR-34a-3p had a significantly higher expression in tumors of NMIBC patients without recurrence (P = 0.0155). Decreased expression of miR-34a-3p was associated with significantly shorter recurrence-free survival (P = 0.009). Cox regression analysis confirmed that miR-34a-3p is an independent biomarker associated with a lower risk of recurrence (hazard ratio (HR) = 0.3184, 95% confidence interval = 0.003-0.681, P = 0.0258). Combination of miR-34a-3p and European Organization for Research and Treatment of Cancer risk score into one predictive model enabled to predict individual risk of recurrence with high statistical significance and analytical performance (P < 0.0001; area under curve = 0.8368; sensitivity 83%, and specificity 75%). CONCLUSIONS: Our data suggest that miR-34a-3p is an independent biomarker of NMIBC recurrence and a promising candidate for further independent validations as an additional factor to improve predictive value of European Organization for Research and Treatment of Cancer nomogram.

Genome-wide identification of urinary cell-free microRNAs for non-invasive detection of bladder cancer.

Urinary microRNAs (miRNAs) are emerging as clinically useful tool for early and non-invasive detection of various types of cancer including bladder cancer (BCA). In this study, 205 patients with BCA and 99 healthy controls were prospectively enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays (2578 miRNAs) and candidate miRNAs further validated in independent cohorts using qRT-PCR. Whole-genome profiling identified 76 miRNAs with significantly different concentrations in urine of BCA compared to controls (P < 0.01). In the training and independent validation phase of the study, miR-31-5p, miR-93-5p and miR-191-5p were confirmed to have significantly higher levels in urine of patients with BCA in comparison with controls (P < 0.01). We further established 2-miRNA-based urinary DxScore (miR-93-5p, miR-31-5p) enabling sensitive BCA detection with AUC being 0.84 and 0.81 in the training and validation phase, respectively. Moreover, DxScore significantly differed in the various histopathological subgroups of BCA and decreased post-operatively. In conclusion, we identified and independently validated cell-free urinary miRNAs as promising biomarkers enabling non-invasive detection of BCA.

Management regime is the most important factor influencing ectomycorrhizal species community in Norway spruce forests after windthrow.

Ectomycorrhizal (ECM) fungi, as symbionts of many tree species in temperate forests, are thought to play an important role in forest regeneration processes after large disturbances. Their reaction to different disturbance and management regimes was studied in spruce forests (Lariceto-Piceetum) 10 years after a severe windthrow in the Tatra National Park (Slovak Republic). ECM community structure was compared between different "management types″-cleared area (EXT), area affected by wildfire (FIRE), uncleared area left for natural development (NEX), and mature forest as a control (REF). Based on Illumina sequencing of soil samples, we determined that the percentage of sequences assigned to ECM fungi decreased with increasing disturbance and management intensity (REF → NEX → EXT → FIRE). Similarly, the total number of ECM species per each of ten sampling points per plot (100 ha) differed between managed (EXT-11 species, FIRE-9) and unmanaged (NEX-16, REF-14) treatments. On the other hand, the percentage of sequences belonging to ericoid mycorrhizal fungi increased. Management type significantly influenced the composition of the ECM community, while vegetation and soil characteristics explained less data variation. The ECM species assemblage of the unmanaged site (NEX) was the most similar to the mature forest, while that of the burnt site was the most different. Thelephora terrestris dominated in all treatments affected by windthrow, accompanied by Tylospora fibrillosa (NEX) and Tylospora asterophora (EXT and FIRE). Management regime was also the most important factor affecting ECM species composition on the roots of spruce seedlings assessed by Sanger sequencing.

mRNA decay is regulated via sequestration of the conserved 5'-3' exoribonuclease Xrn1 at eisosome in yeast.

We describe a novel mechanism of mRNA decay regulation, which takes place under the conditions of glucose deprivation in the yeast Saccharomyces cerevisiae. The regulation is based on temporally stable sequestration of the main 5'-3' mRNA exoribonuclease Xrn1 at the eisosome, a plasma membrane-associated protein complex organizing a specialized membrane microdomain. As documented by monitoring the decay of a specific mRNA substrate in time, Xrn1-mediated mRNA degradation ceases during the accumulation of Xrn1 at eisosome, but the eisosome-associated Xrn1 retains its functionality and can be re-activated when released to cytoplasm following the addition of glucose. In cells lacking the eisosome organizer Pil1, Xrn1 does not associate with the plasma membrane and its activity is preserved till the stationary phase. Thus, properly assembled eisosome is necessary for this kind of Xrn1 regulation, which occurs in a liquid culture as well as in a differentiated colony.

Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans.

The plasma membrane of the fungal pathogen Candida albicans forms a protective barrier that also mediates many processes needed for virulence, including cell wall synthesis, invasive hyphal morphogenesis, and nutrient uptake. Because compartmentalization of the plasma membrane is believed to coordinate these diverse activities, we examined plasma membrane microdomains termed eisosomes or membrane compartment of Can1 (MCC), which correspond to ∼200-nm-long furrows in the plasma membrane. A pil1∆ lsp1∆ mutant failed to form eisosomes and displayed strong defects in plasma membrane organization and morphogenesis, including extensive cell wall invaginations. Mutation of eisosome proteins Slm2, Pkh2, and Pkh3 did not cause similar cell wall defects, although pkh2∆ cells formed chains of furrows and pkh3∆ cells formed wider furrows, identifying novel roles for the Pkh protein kinases in regulating furrows. In contrast, the sur7∆ mutant formed cell wall invaginations similar to those for the pil1∆ lsp1∆ mutant even though it could form eisosomes and furrows. A PH-domain probe revealed that the regulatory lipid phosphatidylinositol 4,5-bisphosphate was enriched at sites of cell wall invaginations in both the sur7∆ and pil1∆ lsp1∆ cells, indicating that this contributes to the defects. The sur7∆ and pil1∆ lsp1∆ mutants displayed differential susceptibility to various types of stress, indicating that they affect overlapping but distinct functions. In support of this, many mutant phenotypes of the pil1∆ lsp1∆ cells were rescued by overexpressing SUR7 These results demonstrate that C. albicans eisosomes promote the ability of Sur7 to regulate plasma membrane organization.