Microcythemia from anemic hypoxia and normal erythropoiesis in the newt

Specimens of the newt, Triturus cristatus carnifex (Laurenti), rendered totally anemic, restore erythron by cyclic waves of erythropoietic activity that alternate with intervals of stasis. Hemolysis is obtained by administering 25 mg/liter of acetylphenylhydrazine in the breeding water for 36 h. The first cycle of erythropoietic activity produces microcytes, which have completely differentiated by 8 weeks after treatment. However, if the animals are raised in a hyperbaric chamber at a pressure of 1.5 atmospheres, in order to compensate for hypoxia, normocytes are produced. In both cases the hematocrit and hematic concentration of hemoglobin reach analogous values, so microcythemia appears to be the only effect of hypoxia. The hemoglobin, hematocrit values, and normocyte counts in hyperbaric animals are about one-half those of the controls newts. These data, together with those on the life span of red blood cells (RBC) and time span between two successive erythropoietic cycles (2 months and 1 month, respectively), indicate that the newts normally keep only two sets (one new, one old) of RBC in circulation, whose approximate parameters can be defined as RBC count: 60,000/mm3, hematocrit: 17%, and hemoglobin: 5.4 g/100 ml.     

Laser scanning cytometry (LCS) allows detailed analysis of the cell cycle in PI stained human fibroblasts (TIG-7)

We have demonstrated a method for the in situ determination of the cell cycle phases of TIG-7 fibroblasts using a laser scanning cytometer (LSC) which has not only a function equivalent to flow cytometry (FCM) but also has a capability unique in itself. LSC allows a more detailed analysis of the cell cycle in cells stained with propidium iodide (PI) than FCM. With LSC it is possible to discriminate between mitotic cells and G₂ cells, between post-mitotic cells and G₁ cells, and between quiescent cells and cycling cells in a PI fluorescence peak (chromatin condensation) vs. fluorescence value (DNA content) cytogram for cells stained with PI. These were amply confirmed by experiments using colcemid and adriamycin. We were able to identify at least six cell subpopulations for PI stained cells using LSC; namely G₁, S, G₂, M, postmitotic and quiescent cell populations. LSC analysis facilitates the monitoring of effects of drugs on the cell cycle.     

Intermittent erythropoiesis in anemic newts

A group of 88 newts, Triturus cristatus carnifex (Laurenti), was rendered totally anemic by administering acetylphenylhydrazine (APH) in the breeding water for 48 h at a concentration of 25 mg/liter. The course of erythron restoration was followed for 5 months, sacrificing four specimens per week and analyzing the blood and spleen hemopoietic tissue. The return to the normal values of the red blood cell count occurred through marked increases in concentration at fairly regular intervals, which is best explained by a discontinuous, rhythmic erythropoiesis. This fact is strictly correlated with the intermittent mitotic activity observed in the spleen and with the periodic appearance of large quantities of immature elements in the blood smears. The APH-induced synchronization of newt erythropoietic activity revealed the approximate length of each erythropoietic cycle to be 4 to 5 weeks and the erythropoietic life span to be 50 to 60 days.     

Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

Background  

The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.     

Enzymatic basis for sialyl-Tn expression in human colon cancer cells

Sialyl-Tn antigen (SA2-6 GalNAc-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells and its expression correlates with a poor prognosis in patients with colorectal and other adenocarcinomas. To understand the enzymatic basis of sialyl-Tn (STn) antigen expression, we used two clonal cell lines, LSB and LSC, derived from LS174T human colonic cancer cells. LSC cells express only the truncated carbohydrate antigen Tn (GalNAc-Ser/Thr) and sialyl-Tn on their mucin molecules, whereas LSB cells express elongated oligosaccharide chains. Both cell lines demonstrated similar activities of glycosyltransferases involved in the biosynthesis of elongated and terminal structures of complex O-glycans. However, LSC cells were unable to synthesize core 1 (Gal1-3GalNAc-) because the ubiquitous enzyme activity of UDP-Gal:GalNAc-R 3-Gal-transferase (core 1 3-Gal-transferase) was lacking. Core 1 3-Gal-transferase could not be reactivated in LSC cells by treatment with sodium butyrate or by in vivo growth of LSC cells in nude mice. In contrast, LSB cells were able to synthesize and process core 1 and core 2 (GlcNAc1-6 (Gal1-3) GalNAc-). LSC cells represent the first example of a non-hematopoietic cell line which lacks core 1 3-Gal-transferase activity. The lack of core 1 3-Gal-transferase in LSC cells explains why they are incapable of forming the common mucin O-glycan core structures and are committed to synthesizing the short Tn and STn oligosaccharides. These findings suggest that the activity of core 1 3-Gal-transferase is an important determinant of the STn phenotype of colon cancer cells.     

Entrainment of the circatidal rhythm of the estuarine amphipod Coroph1um volutator (pallas) to non-tidal cycles of inundation and exposure in the laboratory

The endogenous rhythm of swimming activity shown by the estuarine amphipod Corophium volutator (Pallas) has been studied in animals subjected to periodic inundation under controlled laboratory conditions. A rhythm of circatidal frequency which persisted under constant conditions was recorded in animals entrained to cycles of 8-, 12- and 24-h periods, whereas animals entrained to 6-h cycles appeared to follow the entraining regimen directly, although the free running rhythm was less distinct. Cycles of 4-h period failed to induce rhythmicity. The results support the hypothesis that the endogenous oscillator has a circatidal frequency.

The period for which the animals are submerged is important with regard to entrainment but the effective parameters of the imposed regimen are incompletely understood in this respect.     

Normal Hematopoietic Stem Cells within the AML Bone Marrow Have a Distinct and Higher ALDH Activity Level than Co-Existing Leukemic Stem Cells

Persistence of leukemic stem cells (LSC) after chemotherapy is thought to be responsible for relapse and prevents the curative treatment of acute myeloid leukemia (AML) patients. LSC and normal hematopoietic stem cells (HSC) share many characteristics and co-exist in the bone marrow of AML patients. For the development of successful LSC-targeted therapy, enabling eradication of LSC while sparing HSC, the identification of differences between LSC and HSC residing within the AML bone marrow is crucial. For identification of these LSC targets, as well as for AML LSC characterization, discrimination between LSC and HSC within the AML bone marrow is imperative. Here we show that normal CD34+CD38– HSC present in AML bone marrow, identified by their lack of aberrant immunophenotypic and molecular marker expression and low scatter properties, are a distinct sub-population of cells with high ALDH activity (ALDH^bright). The ALDH^bright compartment contains, besides normal HSC, more differentiated, normal CD34+CD38+ progenitors. Furthermore, we show that in CD34-negative AML, containing solely normal CD34+ cells, LSC are CD34– and ALDH^low. In CD34-positive AML, LSC are also ALDH^low but can be either CD34+ or CD34–. In conclusion, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that LSC have lower ALDH activity than the CD34+CD38– HSC that co-exist with these LSC in the AML bone marrow. Our findings form the basis for combined functionally and immunophenotypically based identification and purification of LSC and HSC within the AML bone marrow, aiming at development of highly specific anti-LSC therapy.     

Transient erythropoietic spleen colonies: effects of erythropoietin in normal and genetically anemic W/Wv mice.

Properties of the cells (TE-CFU) that give rise within four to six days to transient endogenous erythropoietic spleen colonies in irradiated mice have been investigated. The results obtained indicate that (1) erythropoietic maturation within such colonies is highly erythropoietin-dependent, (2) the population size of TE-CFU is not erythropoietin-dependent, (3) initial exposure to a high dose of erythropoietin followed by continuing exposure to lower doses is required for maximal efficiency of colony formation by TE-CFU, (4) successful transplantation of TE-CFU has not been achieved, but they appear among the progeny of transplanted hemopoietic cells, (5) TE-CFU are defective in mice of genotype W/Wv. These findings are consistent with the view that the TE-CFU assay detects a class of early erythropoietin-sensitive progenitor cells committed to erythropoietic diffferentiation, rather than "abortive" colony formation by pluripotent stem cells.     

气候变化背景下贵州省倒春寒灾害时空演变特征

基于1959-2007年贵州省19个气象台站的逐日平均气温资料,结合倒春寒强度指数指标和灾害等级划分标准,从倒春寒发生频率、站次比、年代际变化、气候突变、周期变化等方面,分析了贵州省倒春寒的时空演变特征.结果表明:1959--2007年,研究区无倒春寒发生的频率最大,其次为重级以上倒春寒,中级和轻级倒春寒的发生频率接近;在全球变暖背景下,研究期间贵州省发生中级倒春寒的站次比变化最明显,其气候倾向率达1.4%·(10a)-1,而无倒春寒、轻级和重级以上倒春寒的站次比则略微减少;贵州省倒春寒强度在20世纪90年代最强,20世纪80年代次强,2000-2007年最弱,20世纪70年代次弱,20世纪60年代居中;研究期间贵州省西部和西北部高海拔地区、中部和北部地区的倒春寒强度呈增强趋势,而东部、南部地区的倒春寒强度呈略微降低的趋势;贵州省西部、西北部、中部和北部地区的倒春寒强度在1975年发生由低值向高值的突变;贵州省倒春寒存在明显的周期波动特征,年际周期以2～4年为主,年代际周期以13～15年和27～29年为主.     

Synchronization in chains of pacemaker cells by phase resetting action potential effects

Interactions between pacemaker cells in a chain were calculated according to a "phase-reset" model. It is based on effects of action potentials in the cells on the cycle lengths of neighbouring cells. These effects were defined for each cell by a latency-phase curve (LPC), giving the latency time (L) until the onset of the next action potential in that cell, as a function of the phase (phi) at which a neighbour cell fired an action potential. Neighbour cells with simultaneous action potentials did not influence each others cycle length. We investigated how stable synchronization depends on the shape of the LPC's of the pacemaker cells and on chain length. Three types of interactive behaviour were distinguished. First, anti-phase synchrony, in which neighbouring cells fired with large phase differences with respect to the synchronized period Ps. Second, asynchrony, in which the periods of the cells did not become equal and constant. Third, in-phase synchrony, in which the phase differences between the neighbouring cells were zero or much smaller than the synchronized period Ps, depending on the differences between the intrinsic periods. Asynchrony and anti-phase synchrony may be seen as cardiophysiological arrhythmias, while in-phase synchrony represents the physiological type of synchrony in the heart. In-phase synchrony appeared to be strongly favoured by LPC's, which have a no-effect (refractory) part at early phases, a lengthened latency (or phase delay) part at intermediate phases and a shortened latency (or phase advance) part at late phases in the cycle. Such LPC-shapes are commonly found in preparations of cardiac pacemaker cells. When the pacemaker cells were identical, the synchronized period Ps during in-phase synchrony was equal to their intrinsic period P*i. For different intrinsic periods, Ps was equal to the intrinsic period of the fastest cell if the LPC's contained a sufficiently long initial no-effect period at early phases and a shortened latency part at late phases. When, on the other hand, such cell chains had a linear gradient in their intrinsic periods, "action potentials" started from the fast end and traveled along the chain. The propagation of an action potential wave slowed down as it reached the slower cells. When the gradient in the intrinsic periods was too steep, only the intrinsically fast end of the chain developed synchrony.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a third pathway for arachidonic acid mobilization and prostaglandin production in activated P388D1 macrophage-like cells

Previous studies have demonstrated that P388D(1) macrophages are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in two temporally distinct phases. The first phase is triggered by platelet-activating factor within minutes, but needs the cells to be previously exposed to bacterial lipopolysaccharide (LPS) for periods up to 1 h. It is thus a primed immediate phase. The second, delayed phase occurs in response to LPS alone over long incubation periods spanning several hours. Strikingly, the effector enzymes involved in both of these phases are the same, namely the cytosolic group IV phospholipase A(2) (cPLA(2)), the secretory group V phospholipase A(2), and cyclooxygenase-2, although the regulatory mechanisms differ. Here we report that P388D(1) macrophages mobilize AA and produce prostaglandins in response to zymosan particles in a manner that is clearly different from the two described above. Zymosan triggers an immediate AA mobilization response from the macrophages that neither involves the group v phospholipase A(2) nor requires the cells to be primed by LPS. The group VI Ca(2+)-independent phospholipase A(2) is also not involved. Zymosan appears to signal exclusively through activation of the cPLA(2), which is coupled to the cyclooxygenase-2. These results define a secretory PLA(2)-independent pathway for AA mobilization in the P388D(1) macrophages, and demonstrate that, under certain experimental settings, stimulation of the cPLA(2) is sufficient to generate a prostaglandin biosynthetic response in the P388D(1) macrophages.     

Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry

BACKGROUND: Caspase activation is a critical early step in the onset of apoptosis. Cell-permeable fluorogenic caspase substrates have proven valuable in detecting caspase activation by flow cytometry. Nevertheless, detection of early low-level caspase activation has been difficult using conventional area or peak fluorescence analysis by flow cytometry, despite the apparent presence of these cells as observed by microscopy. We describe a method utilizing maximum fluorescence pixel analysis by laser scanning cytometry (LSC) to detect early apoptotic cells. METHODS: The PhiPhiLux-G(1)D(2) caspase 3/7 substrate was used in combination with DNA dye exclusion and annexin V binding to identify several stages of apoptosis in EL4 murine thymoma cells by both traditional flow and LSC. LSC analysis of maximum pixel brightness in individual cells demonstrated an intermediate caspase-low subpopulation not detectable by flow or LSC integral analysis. LSC analysis of caspase activity was then carried out using the larger UMR-106 rat osteosarcoma cell line to determine if this apparent early caspase activity could be correlated with localized, punctate caspase activity observed by microscopy. RESULTS: The caspase-low subpopulation found in apoptotic EL4 cells was also observable in UMR-106 cells. Relocation to cells with low fluorescence due to caspase activity and subsequent examination by microscopy demonstrated that these latter cells indeed show punctate, highly localized caspase activation foci that might represent an early stage in caspase activation. CONCLUSIONS: Cells with low-level, localized caspase expression can be detected using maximum pixel analysis by LSC. This methodology allows an early step of apoptotic activation to be resolved for further analysis.     

Endothelial cells create a hematopoietic inductive microenvironment preferential to erythropoiesis in the mouse spleen

The spleen is an erythropoietic organ in mouse. To reconstruct a microenvironment essential for erythropoiesis in vitro, the stroma (MSS31) cell line had been established from a newborn mouse spleens. MSS31 cells exhibited properties of endothelial cells: (a) the cells showed the activity to uptake acetylated low-density lipoprotein (Ac-LDL) and (b) the cells can form a capillarylike structure by a phenotypic modulation in collagen matrices. MSS31 cells selectively supported the proliferation and differentiation of the erythroid progenitor cells by direct cell-to-cell contact in a semisolid medium in the presence of erythropoietin. These layers also supported erythrocyte maturation and enucleation of erythroblasts. This suggests that spleen endothelial cells are a new type of stromal cell with erythropoietic stimulation activity and may have a critical function in the hemopoietic inductive microenvironment of the mouse spleen.     

Autoregulation of the electrogenic sodium pump

The dependence of electrogenic sodium pump activity on changes in the cell volume of Helix pomatia neurons with different levels of intracellular sodium ion concentration was studied. Hypertonic solutions caused hyperpolarization of the membrane and increased membrane resistance in cells with a low sodium content (low-sodium cells; LSC). The activity of the electrogenic sodium pump in hypertonic solutions was increased compared to the activity in hypotonic solutions in LSC and decreased in cells with a high sodium content (high-sodium cells; HSC). The concentration of ouabain which led to maximal inhibition of active 22Na efflux from the neurons was 10(-4) M. Lower concentrations of ouabain (10(-8) M and lower) did not inhibit the sodium pump but stimulated it. The swelling of neurons in hypotonic solutions was accompanied by an increase in the number of binding sites for ouabain, while shrinking in hypertonic solutions led to the opposite effect--a decrease in binding sites. An increase in the number of binding sites also took place in normal isotonic potassium-free solutions compared with normal Ringer's solution. Two saturable components of ouabain binding were detectable in all solutions examined. gamma-Aminobutyric acid (GABA) and acetylcholine (ACh) increased the number of ouabain binding sites on the membrane. The results suggest that there are two opposite mechanisms by which cell volume changes can modulate the pump activity. One of them depends on the intracellular sodium ion concentration and causes pump activation in hypertonic solutions in LSC and saturation in HSC, while a second mechanism mediates the activating effect of cell swelling on the sodium pump in HSC. In addition, there may be a negative feedback between the pump activity and the number of functioning pump units in the membrane.     

Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture

When embryonic stem cells are cultured directly in semisolid media (methyl cellulose), they proliferate and differentiate to generate colonies known as embryoid bodies (EBs). These EBs consist of differentiated cells from a number of lineages including those of the hematopoietic system. Following 10 days of culture in the presence of 10% fetal calf serum, more than 40% of all EBs from three different ES cell lines, CCEG2, D3 and SQ1.2S8 contained visible erythropoietic cells (i.e. red with hemoglobin). Beta H1 (z globin) mRNA is detectable in EBs within 5 days of differentiation, whilst beta(maj)-globin RNA appears by day 6. In the presence of erythropoietin (Epo), the frequency of EBs with erythropoietic activity increases to greater than 60%; Epo also prolongs this erythropoietic activity. Interleukin-3 (IL-3) does not significantly increase the frequency of EBs that contain erythroid cells, but increases slightly the number of erythropoietic cells associated with them. In the presence of IL-3, in addition to cells of the erythroid lineage, macrophages, mast cells and in some instances neutrophils are found within differentiating EBs. The development of macrophages is significantly enhanced by the addition of IL-3 alone or in combination with IL-1 and M-CSF or GM-CSF. When well-differentiated EBs are allowed to attach onto tissue-culture plates and grown in the presence of IL-3, a long-term output of cells from the mast cell lineage is observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of serotonin and acetylcholine on the electrical activity of isolated cortex in the rabbit

Neuronal isolation of the rabbit's cerebral hemisphere shifts the EEG spectrum in direction of slower processes. Application of acetylcholine on the cortex elicits EEG activation and appearance of the theta-rhythm. Initially serotonin application is accompanied by the appearance of the theta-rhythm periods; in the process of subsequent administration of the drug these periods are gradually substituted by slow delta-waves. Combined application of serotonin and acetylcholine on isolated cortex elicits bursts of high-amplitude activity, abruptly substituted by "silence" phases. In contrast to the intact cortex where serotonin elicited prolonged and rhythmic alternation on EEG of phases of high-amplitude activity and of "silence" periods, in the isolated cortex the bursts of activity of about 1 min duration appeared only after application of the acetylcholine to serotonin-saturated cortex. Repeated phases of activation were either absent or were of short duration and extinguished rapidly.     

Gates and oscillators: a network model of the brain clock

The suprachiasmatic nuclei (SCN) control circadian oscillations of physiology and behavior. Measurements of electrical activity and of gene expression indicate that these heterogeneous structures are composed of both rhythmic and nonrhythmic cells. A fundamental question with regard to the organization of the circadian system is how the SCN achieve a coherent output while their constituent independent cellular oscillators express a wide range of periods. Previously, the consensus output of individual oscillators had been attributed to coupling among cells. The authors propose a model that incorporates nonrhythmic "gate" cells and rhythmic oscillator cells with a wide range of periods, that neither requires nor excludes a role for interoscillator coupling. The gate provides daily input to oscillator cells and is in turn regulated (directly or indirectly) by the oscillator cells. In the authors' model, individual oscillators with initial random phases are able to self-assemble so as to maintain cohesive rhythmic output. In this view, SCN circuits are important for self-sustained oscillation, and their network properties distinguish these nuclei from other tissues that rhythmically express clock genes. The model explains how individual SCN cells oscillate independently and yet work together to produce a coherent rhythm.     

In-vivo myometrial electrical activity in the cyclic mare

Uterine electromyography was performed by means of chronically implanted surface electrodes in 3 Pony mares during spontaneous oestrous cycles and following luteolysis induced by a prostaglandin analogue (fluprostenol). Three distinct patterns were recognized during the oestrous cycle. (1) During oestrus well defined phases of activity with closely grouped high-amplitude spikes were separated by long periods (10-45 min) of complete inactivity. (2) During dioestrus more diffuse phases of activity with low-amplitude spikes were separated by variable periods of relative inactivity. (3) During luteolysis, short and frequently occurring phases of activity were propagated between the two electrodes on one uterine horn; a similar pattern also occurred between 1 and 3 h after injection of fluprostenol. Peripheral plasma progesterone, but not total inconjugated oestrogen, concentrations were closely related to characteristics of the myographic activity during the cycle. Insemination during oestrus and injection of fluprostenol during dioestrus caused a marked and prolonged increase in myometrial electrical activity. Almost any non-specific environmental stimulus, including entry by palpation of the genital tract per rectum and vaginoscopic examination, but these were of brief duration and the normal resting pattern of activity was quickly re-established after completion of the manipulations.     

Leptin secreted from testicular microenvironment modulates hedgehog signaling to augment the endogenous function of Leydig cells

Although testosterone deficiency (TD) may be present in one out of five men 40 years or older, the factors responsible for TD remain largely unknown. Leydig stem cells (LSCs) differentiate into adult Leydig cells (ALC) and produce testosterone in the testes under the pulsatile control of luteinizing hormone (LH) from the pituitary gland. However, recent studies have suggested that the testicular microenvironment (TME), which is comprised of Sertoli and peritubular myoid cells (PMC), plays an instrumental role in LSC differentiation and testosterone production under the regulation of the desert hedgehog signaling pathway (DHH). It was hypothesized that the TME releases paracrine factors to modulate LSC differentiation. For this purpose, cells (Sertoli, PMCs, LSCs, and ALCs) were extracted from men undergoing testis biopsies for sperm retrieval and were evaluated for the paracrine factors in the presence or absence of the TME (Sertoli and PMC). The results demonstrated that TME secretes leptin, which induces LSC differentiation and increases testosterone production. Leptin’s effects on LSC differentiation and testosterone production, however, are inversely concentration-dependent: positive at low doses and negative at higher doses. Mechanistically, leptin binds to the leptin receptor on LSCs and induces DHH signaling to modulate LSC differentiation. Leptin-DHH regulation functions unidirectionally insofar as DHH gain or loss of function has no effect on leptin levels. Taken together, these findings identify leptin as a key paracrine factor released by cells within the TME that modulates LSC differentiation and testosterone release from mature Leydig cells, a finding with important clinical implications for TD.Subject terms: Stem-cell differentiation, Translational research     

Centennial- to decadal scale environmental shifts in and around Lake Pannon (Vienna Basin) related to a major Late Miocene lake level rise

A detailed ultra-high-resolution analysis of a 37-cm-long core of Upper Miocene lake sediments of the long-lived Lake Pannon has been performed. Despite a general stable climate at c. 11-9 Ma, several high-frequency oscillations of the paleoenvironments and depositional environments are revealed by the analysis over a short time span of less than 1000 years. Shifts of the lake level, associated with one major 3rd order flooding are reflected by all organisms by a cascade of environmental changes on a decadal scale. Within a few decades, the pollen record documents shifting vegetation zones due to the landward migration of the coast; the dinoflagellate assemblages switch towards "offshore-type" due to the increasing distance to the shore; the benthos is affected by low oxygen conditions due to the deepening. This general trend is interrupted by smaller scale cycles, which lack this tight interconnection. Especially, the pollen data document a clear cyclicity that is expressed by iterative low pollen concentration events. These "negative" cycles are partly reflected by dinoflagellate blooms suggesting a common trigger-mechanism and a connection between terrestrial environments and surface waters of Lake Pannon. The benthic fauna of the core, however, does not reflect these surface water cycles. This forcing mechanism is not understood yet but periodic climatic fluctuations are favoured as hypothesis instead of further lake level changes. Short phases of low precipitation, reducing pollen production and suppressing effective transport by local streams, might be a plausible mechanism. This study is the first hint towards solar activity related high-frequency climate changes during the Vallesian (Late Miocene) around Lake Pannon and should encourage further ultra-high-resolution analyses in the area.