Comparison of the distribution and phenology of Arctic Mountain plants between the early 20th and 21st centuries.

Arctic plants are adapted to climatic variability, but their long-term responses to warming remain unclear. Responses may occur by range shifts, phenological adjustments in growth and reproduction, or both. Here, we compare distribution and phenology of 83 arctic and boreal mountain species, sampled identically in the early 20th (1917-1919) and 21st centuries (2017-2018) from a region of northern Sweden that has warmed significantly. We test two compensatory hypotheses to high-latitude warming-upward shifts in distribution, and earlier or extended growth and reproduction. For distribution, we show dramatic upward migration by 69% of species, averaging 6.1 m per decade, especially boreal woodland taxa whose upward expansion has reduced arctic montane habitat by 30%. Twenty percent of summit species showed distributional shifts but downward, especially moisture-associated snowbed flora. For phenology, we detected wide inter-annual variability in the onset of leafing and flowering in both eras. However, there was no detectable change in growing-season length, relating to two mechanisms. First, plot-level snow melt data starting in 1917 demonstrated that melt date, rather than vernal temperatures, better predicts plant emergence, with snow melt influenced by warmer years having greater snowfall-warmer springs did not always result in earlier emergence because snowbeds can persist longer. Second, the onset of reproductive senescence between eras was similar, even when plant emergence was earlier by a month, possibly due to intensified summer heat stress or hard-wired 'canalization' where senescence occurs regardless of summer temperature. Migrations in this system have possibly buffered arctic species against displacement by boreal expansion and warming, but ongoing temperature increases, woody plant invasion, and a potential lack of flexibility in timing of senescence may foreshadow challenges.

Identification of biomarkers of human skin ageing in both genders. Wnt signalling - a label of skin ageing?

The goal of our work has been to investigate the mechanisms of gender-independent human skin ageing and examine the hypothesis of skin being an adequate model of global ageing. For this purpose, whole genome gene profiling was employed in sun-protected skin obtained from European Caucasian young and elderly females (mean age 26.7±4 years [n1 = 7] and 70.75±3.3 years [n2 = 4], respectively) and males (mean age 25.8±5.2 years [n3 = 6] and 76±3.8 years [n4 = 7], respectively) using the Illumina array platform. Confirmation of gene regulation was performed by real-time RT-PCR and immunohistochemistry. 523 genes were significantly regulated in female skin and 401 genes in male skin for the chosen criteria. Of these, 183 genes exhibited increased and 340 decreased expression in females whereas 210 genes showed increased and 191 decreased expression in males with age. In total, 39 genes were common in the target lists of significant regulated genes in males and females. 35 of these genes showed increased (16) or decreased (19) expression independent of gender. Only 4 overlapping genes (OR52N2, F6FR1OP2, TUBAL3 and STK40) showed differential regulation with age. Interestingly, Wnt signalling pathway showed to be significantly downregulated in aged skin with decreased gene and protein expression for males and females, accordingly. In addition, several genes involved in central nervous system (CNS) ageing (f.i. APP, TAU) showed to be expressed in human skin and were significanlty regulated with age. In conclusion, our study provides biomarkers of endogenous human skin ageing in both genders and highlight the role of Wnt signalling in this process. Furthermore, our data give evidence that skin could be used as a good alternative to understand ageing of different tissues such as CNS.

Review: cerebral microvascular pathology in ageing and neurodegeneration.

This review of age-related brain microvascular pathologies focuses on topics studied by this laboratory, including anatomy of the blood supply, tortuous vessels, venous collagenosis, capillary remnants, vascular density and microembolic brain injury. Our studies feature thick sections, large blocks embedded in celloidin, and vascular staining by alkaline phosphatase. This permits study of the vascular network in three dimensions, and the differentiation of afferent from efferent vessels. Current evidence suggests that there is decreased vascular density in ageing, Alzheimer's disease and leukoaraiosis, and cerebrovascular dysfunction precedes and accompanies cognitive dysfunction and neurodegeneration. A decline in cerebrovascular angiogenesis may inhibit recovery from hypoxia-induced capillary loss. Cerebral blood flow is inhibited by tortuous arterioles and deposition of excessive collagen in veins and venules. Misery perfusion due to capillary loss appears to occur before cell loss in leukoaraiosis, and cerebral blood flow is also reduced in the normal-appearing white matter. Hypoperfusion occurs early in Alzheimer's disease, inducing white matter lesions and correlating with dementia. In vascular dementia, cholinergic reductions are correlated with cognitive impairment, and cholinesterase inhibitors have some benefit. Most lipid microemboli from cardiac surgery pass through the brain in a few days, but some remain for weeks. They can cause what appears to be a type of vascular dementia years after surgery. Donepezil has shown some benefit. Emboli, such as clots, cholesterol crystals and microspheres can be extruded through the walls of cerebral vessels, but there is no evidence yet that lipid emboli undergo such extravasation.

Comparative proteome and transcriptome analyses of embryonic stem cells during embryoid body-based differentiation.

Gene expression analyses of embryonic stem cells (ESCs) will help to uncover or further define signaling pathways and molecular mechanisms involved in the maintenance of self-renewal and pluripotency. We employed a 2-DE-based proteomics approach to analyze human ESC line, Royan H5, in undifferentiated cells and different stages of spontaneous differentiation (days 3, 6, 12, and 20) by embryoid body formation. Out of 945 proteins reproducibly detected on gels, the expression of 96 spots changed during differentiation. Using MS, 87 ESC-associated proteins were identified including several proteins involved in cell proliferation, cell apoptosis, transcription, translation, mRNA processing, and protein folding. Transcriptional changes accompanying differentiation of Royan H5 were also analyzed using microarrays. We developed a comprehensive data set that shows the use of human ESC lines in vitro to mimic gastrulation and organogenesis. Our results showed that proteomics and transcriptomics data are complementary rather than duplicative. Although regulation of many genes during differentiation were observed only at transcript level, modulation of several proteins was revealed only by proteome analysis.

Oct-4 regulates the expression of Stella and Foxj2 at the Nanog locus: implications for the developmental competence of mouse oocytes.

BACKGROUND: Our knowledge of what determines the mammalian oocyte developmental competence is meagre. By comparing the transcriptional profiles of developmentally competent surrounded nucleolus (SN) and incompetent not surrounded nucleolus (NSN) mouse MII oocytes, we recently demonstrated that Oct-4 and Stella are key factors in the establishment of the oocytes' developmental competence. METHODS: Using RT-PCR, microarray and immunocytochemistry assays, we analysed expression of genes and proteins in oocytes isolated throughout folliculogenesis and classified based on their SN- or NSN-type of chromatin organization. RESULTS: We show that: (1) Oct-4 and Stella are expressed concurrently at the beginning of oocytes' growth and only in SN oocytes; (2) Germ Cell Nuclear Factor is a putative regulator of Oct-4 expression in MII oocytes; (3) the function of Oct-4 is directed at the Nanog locus, regulating the expression of Stella and Foxj2. CONCLUSIONS: (1) A number of factors that act upstream and downstream of Oct-4 emerge as candidate players in the acquisition of the oocyte's developmental competence; (2) we define molecular markers that identify a specific group of ovarian oocytes (SN) that have a potential to acquire developmental competence; (3) the presence of SN and NSN oocytes in human ovaries extends the interest of these results to the field of human reproduction.

Activation of the immune response is a key feature of aging in mice.

The process of aging is complex involving numerous factors centered on transcriptional changes with advanced age. This study was aimed at elucidating mechanisms involved in mouse aging by conducting both gene expression and biochemical analyses on isolated mouse brain, heart and kidney. The gene expression analysis was not aimed at solely highlighting age-related transcriptional changes but also revealing regulated biological processes, cellular compartments, signaling and metabolic pathways. We have uncovered a conserved increase in the expression of genes mediating immune responses in all the tissues analyzed. In addition, elevated levels of lipid hydroperoxides (LPO)­an indicator of increased levels of radical oxygen species, implicate an oxidative stress-mediated activity of NF-kB signaling. In summary, these results suggest that transcriptional changes are most probably the downstream effect of environmental and endogenous factors constantly affecting the organism during its lifetime. In addition, we propose LPO as a potential biomarker of aging.

Age-related transcriptional changes in gene expression in different organs of mice support the metabolic stability theory of aging.

Individual differences in the rate of aging are determined by the efficiency with which an organism transforms resources into metabolic energy thus maintaining the homeostatic condition of its cells and tissues. This observation has been integrated with analytical studies of the metabolic process to derive the following principle: The metabolic stability of regulatory networks, that is the ability of cells to maintain stable concentrations of reactive oxygen species (ROS) and other critical metabolites is the prime determinant of life span. The metabolic stability of a regulatory network is determined by the diversity of the metabolic pathways or the degree of connectivity of genes in the network. These properties can be empirically evaluated in terms of transcriptional changes in gene expression. We use microarrays to investigate the age-dependence of transcriptional changes of genes in the insulin signaling, oxidative phosphorylation and glutathione metabolism pathways in mice. Our studies delineate age and tissue specific patterns of transcriptional changes which are consistent with the metabolic stability-longevity principle. This study, in addition, rejects the free radical hypothesis which postulates that the production rate of ROS, and not its stability, determines life span.

Maternal Oct-4 is a potential key regulator of the developmental competence of mouse oocytes.

BACKGROUND: The maternal contribution of transcripts and proteins supplied to the zygote is crucial for the progression from a gametic to an embryonic control of preimplantation development. Here we compared the transcriptional profiles of two types of mouse MII oocytes, one which is developmentally competent (MIISN oocyte), the other that ceases development at the 2-cell stage (MIINSN oocyte), with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence. RESULTS: We report that: 1) the transcription factor Oct-4 is absent in MIINSN oocytes, accounting for 2) the down-regulation of Stella, a maternal-effect factor required for the oocyte-to-embryo transition and of which Oct-4 is a positive regulator; 3) eighteen Oct-4-regulated genes are up-regulated in MIINSN oocytes and are part of gene expression networks implicated in the activation of adverse biochemical pathways such as oxidative phosphorylation, mitochondrial dysfunction and apoptosis. CONCLUSION: The down-regulation of Oct-4 plays a crucial function in a sequence of molecular processes that leads to the developmental arrest of MIINSN oocytes. The use of a model study in which the MII oocyte ceases development consistently at the 2-cell stage has allowed to attribute a role to the maternal Oct-4 that has never been described before. Oct-4 emerges as a key regulator of the molecular events that govern the establishment of the developmental competence of mouse oocytes.

The origins of human embryonic stem cells: a biological conundrum.

Human inner cell mass (ICM) cells isolated from in vitro fertilized blastocysts are the progenitor cells used to establish in vitro stable human embryonic stem cells (hESCs) which are pluripotent and self-renew indefinitely. This long-term perpetuation of hESCs in the undifferentiated state is thought to be an in vitro adaptation of the ICM cells. To investigate at the molecular level how hESCs acquired their unique properties, transcriptional profiles of isolated ICM cells and undifferentiated hESCs were compared. We identified 33 genes enriched in the ICM compared to the trophectoderm and hESCs. These genes are involved in signaling cascades (SEMA7A and MAP3K10), cell proliferation (CUZD1 and MS4A7) and chromatin remodeling (H1FOO and HRMT1L4). Furthermore, primordial germ cell-specific genes (SGCA and TEX11) were detected as expressed in the ICM cells and not hESCs. We propose that the transcriptional differences observed between ICM cells and hESCs might be accounted for by adaptive reprogramming events induced by the in vitro culture conditions which are distinct from that of in vitro fertilized blastocysts. hESCs are a distinct cell type lacking in the human embryo but, nonetheless, resemble the ICM in their ability to differentiate into cells representative of the endodermal, ectodermal and mesodermal cell lineages.

Conserved molecular portraits of bovine and human blastocysts as a consequence of the transition from maternal to embryonic control of gene expression.

The present study investigated mRNA expression profiles of bovine oocytes and blastocysts by using a cross-species hybridization approach employing an array consisting of 15,529 human cDNAs as probe, thus enabling the identification of conserved genes during human and bovine preimplantation development. Our analysis revealed 419 genes that were expressed in both oocytes and blastocysts. The expression of 1,324 genes was detected exclusively in the blastocyst, in contrast to 164 in the oocyte including a significant number of novel genes. Genes indicative for transcriptional and translational control (ELAVL4, TACC3) were overexpressed in the oocyte, whereas cellular trafficking (SLC2A14, SLC1A3), proteasome (PSMA1, PSMB3), cell cycle (BUB3, CCNE1, GSPT1), and protein modification and turnover (TNK1, UBE3A) genes were found to be overexpressed in blastocysts. Transcripts implicated in chromatin remodeling were found in both oocytes (NASP, SMARCA2) and blastocysts (H2AFY, HDAC7A). The trophectodermal markers PSG2 and KRT18 were enriched 5- and 50-fold in the blastocyst. Pathway analysis revealed differential expression of genes involved in 107 distinct signaling and metabolic pathways. For example, phosphatidylinositol signaling and gluconeogenesis were prominent pathways identified in the blastocyst. Expression patterns in bovine and human blastocysts were to a large extent identical. This analysis compared the transcriptomes of bovine oocytes and blastocysts and provides a solid foundation for future studies on the first major differentiation events in blastocysts and identification of a set of markers indicative for regular mammalian development.

Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells.

The POU domain transcription factor OCT4 is a key regulator of pluripotency in the early mammalian embryo and is highly expressed in the inner cell mass of the blastocyst. Consistent with its essential role in maintaining pluripotency, Oct4 expression is rapidly downregulated during formation of the trophoblast lineage. To enhance our understanding of the molecular basis of this differentiation event in humans, we used a functional genomics approach involving RNA interference-mediated suppression of OCT4 function in a human ESC line and analysis of the resulting transcriptional profiles to identify OCT4-dependent genes in human cells. We detected altered expression of >1,000 genes, including targets regulated directly by OCT4 either positively (NANOG, SOX2, REX1, LEFTB, LEFTA/EBAF DPPA4, THY1, and TDGF1) or negatively (CDX2, EOMES, BMP4, TBX18, Brachyury [T], DKK1, HLX1, GATA6, ID2, and DLX5), as well as targets for the OCT4-associated stem cell regulators SOX2 and NANOG. Our data set includes regulators of ACTIVIN, BMP, fibroblast growth factor, and WNT signaling. These pathways are implicated in regulating human ESC differentiation and therefore further validate the results of our analysis. In addition, we identified a number of differentially expressed genes that are involved in epigenetics, chromatin remodeling, apoptosis, and metabolism that may point to underlying molecular mechanisms that regulate pluripotency and trophoblast differentiation in humans. Significant concordance between this data set and previous comparisons between inner cell mass and trophectoderm in human embryos indicates that the study of human ESC differentiation in vitro represents a useful model of early embryonic differentiation in humans.

Age-specific hormonal decline is accompanied by transcriptional changes in human sebocytes in vitro.

The importance of hormones in endogenous aging has been displayed by recent studies performed on animal models and humans. To decipher the molecular mechanisms involved in aging we maintained human sebocytes at defined hormone-substituted conditions that corresponded to average serum levels of females from 20 (f20) to 60 (f60) years of age. The corresponding hormone receptor expression was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunocytochemistry. Cells at f60 produced significantly lower lipids than at f20. Increased mRNA and protein levels of c-Myc and increased protein levels of FN1, which have been associated with aging, were detected in SZ95 sebocytes at f60 compared to those detected at f20 after 5 days of treatment. Expression profiling employing a cDNA microarray composed of 15 529 cDNAs identified 899 genes with altered expression levels at f20 vs. f60. Confirmation of gene regulation was performed by real-time RT-PCR. The functional annotation of these genes according to the Gene Ontology identified pathways related to mitochondrial function, oxidative stress, ubiquitin-mediated proteolysis, cell cycle, immune responses, steroid biosynthesis and phospholipid degradation - all hallmarks of aging. Twenty-five genes in common with those identified in aging kidneys and several genes involved in neurodegenerative diseases were also detected. This is the first report describing the transcriptome of human sebocytes and its modification by a cocktail of hormones administered in age-specific levels and provides an in vitro model system, which approximates some of the hormone-dependent changes in gene transcription that occur during aging in humans.

Primary differentiation in the human blastocyst: comparative molecular portraits of inner cell mass and trophectoderm cells.

The primary differentiation event during mammalian development occurs at the blastocyst stage and leads to the delineation of the inner cell mass (ICM) and the trophectoderm (TE). We provide the first global mRNA expression data from immunosurgically dissected ICM cells, TE cells, and intact human blastocysts. Using a cDNA microarray composed of 15,529 cDNAs from known and novel genes, we identify marker transcripts specific to the ICM (e.g., OCT4/POU5F1, NANOG, HMGB1, and DPPA5) and TE (e.g., CDX2, ATP1B3, SFN, and IPL), in addition to novel ICM- and TE-specific expressed sequence tags. The expression patterns suggest that the emergence of pluripotent ICM and TE cell lineages from the morula is controlled by metabolic and signaling pathways, which include inter alia, WNT, mitogen-activated protein kinase, transforming growth factor-beta, NOTCH, integrin-mediated cell adhesion, phosphatidylinositol 3-kinase, and apoptosis. These data enhance our understanding of the first step in human cellular differentiation and, hence, the derivation of both embryonic stem cells and trophoblastic stem cells from these lineages.

Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays.

BACKGROUND: Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe. RESULTS: As a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs. CONCLUSIONS: Results of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different species.

A three-dimensional study of brain string vessels using celloidin sections stained with anti-collagen antibodies.

The purpose of this study was to explain the morphology and significance of string vessels in human brains. Brain slices (1.5 cm thick) were embedded in celloidin, sections cut at 100 microm and stained with antibody to collagen IV. A second component of the study was a 3-D rotational study for which we used sections stained with propidium iodide for cell nuclei and anti-collagen stain for blood vessel basement membranes. The materials consisted of brain from two infants at 28 and 35 weeks gestation, two term infants at 20 days and 3 months, one 5 years old, and 3 adults aged 25, 57, and 84 years. String vessels were counted in at least six fields of deep white matter using a 10x objective and the counts averaged and expressed as string vessels per cubic mm. The 3-D rotational study using confocal microscopy was designed to find nuclei in string vessels. The least number of string vessels were present in the premature infant. All others had comparably similar numbers of string vessels except the two term-born infants in whom there was a 3-5-fold increase. However, the two brains had other pathologic lesions, which could affect the counts. In normal brains, string vessels appear as a singe line of stain and usually connect two arterioles or capillaries. They can form loops and occasionally a string vessel may continue into a normal capillary. String vessels have rare nuclei. Our study indicates that string vessels are present in utero, increase in number and are present throughout life. Their exact nature remains unexplained. They apparently do not represent age-related acquired atrophy of capillaries because they are present at all ages and do not progressively increase with normal aging. This technique appears suitable for the study of large number of string vessels.

Ischemia increases prostaglandin H synthase-2 levels in retina and visual cortex in piglets.

BACKGROUND: Ischemia increases levels of prostaglandin H synthase-2 (PGHS-2) in neonatal brain and cerebral vasculature, but effects on the developing visual system are unknown. We examined the effects of ischemia on PGHS-2 mRNA and protein levels in the retina and visual cortex in anesthetized piglets. METHODS: Ten minutes of complete retinal and brain ischemia was induced by increasing intracranial pressure. After 2-12 h of reperfusion, samples of retina and visual cortex were collected for determinations of levels of PGHS-2 mRNA (RNase protection assay) or protein (immunohistochemistry and western blotting). Tissues also were obtained from control animals. RESULTS: Levels of PGHS-2 mRNA were undetectable in control animals but showed a dramatic increase at 2-4 h in the cortex and retina in animals exposed to ischemia. Detectable but limited PGHS-2 immunoreactivity (IR) was present in the retina and visual cortex from control animals. In piglets not subjected to ischemia, PGHS-2 IR was localized mainly to the outer limiting membrane and to the Muller cells. Ischemia induced a marked increase in PGHS-2 IR in the neural retina, with the greatest increase in the photoreceptor layer. PGHS-2 levels in whole retina also increased at 8 h after ischemia. In the intact visual cortex PGHS-2 IR was evident in layers II and V. Ischemia increased the intensity of IR in layers II/III as well as layer V. CONCLUSIONS: Detectable amounts of PGHS-2 protein are present in the piglet retina and visual cortex under normal conditions, but levels are markedly increased 8-12 h after ischemic stress. Enhanced PGHS-2 levels after ischemic stress may contribute to delayed pathological changes of the visual system in the neonate.

Cerebrovascular pathology in Alzheimer's disease and leukoaraiosis.

A high percentage of patients with Alzheimer's disease (AD) show evidence of white matter degeneration known as leukoaraiosis (LA), which is due to chronic ischemia. We found that the periventricular veins tend to become occluded by multiple layers of collagen in the vessel walls in the elderly. This collagen deposition is particularly excessive in LA lesions. Therefore, it is present in the brains of many AD patients, along with other ischemia-causing cerebrovascular pathology. We found evidence that there is severe loss of oligodendrocytes in LA, due to extensive apoptosis. No evidence of inflammation was found in the LA lesions. In thick celloidin sections of AD brain, we have obtained detailed 3D views of small (early) deposits of amyloid (stained with beta-amyloid antibody) around capillaries (stained with collagen IV antibody).

Apoptosis in leukoaraiosis.

We report a case of leukoaraiosis that was studied for apoptosis. In the neuropil, the number of cells that showed DNA fragmentation was 2.5 times as great in the area of leukoaraiosis as in the adjacent white matter (P = .004) and 25 times as great as in the nearby cortex (P < .001). Our findings suggest that apoptosis, predominantly of oligodendrocytes, is involved in the pathogenesis of leukoaraiosis. Within the area of leukoaraiosis, we also found numerous small veins that were partially occluded by severe collagenous thickening of the vessel walls. This collagenosis may have contributed to or resulted from chronic ischemia in that area.

Effects of ischemia on prostaglandin H synthase-2 expression in piglet choroid plexus.

We examined effects of ischemia on expression of prostaglandin H synthase-1 (PGHS-1) and prostaglandin H synthase-2 (PGHS-2) in piglet choroid plexus. Ten minutes of ischemia was induced by increasing intracranial pressure. Whole choroid plexus was removed and fixed and/or frozen after 1, 2, 4, and 8 h of recovery from anoxic stress. In addition, tissues were obtained from untreated animals or from time control animals. Tissues were analyzed for mRNA, using RNase protection assays, and for proteins, using immunohistochemical approaches. Limited, but detectable PGHS-2 immunoreactivity was present in choroid plexus under normal conditions, and there was no difference between time-control and non-treated animals. Further, PGHS-2 mRNA increased by 2-4 h after ischemia, and enhanced immunoreactivity for PGHS-2 was present at 8 h after ischemia. Enhanced immunoreactivity for PGHS-2 was present in vascular endothelial cells as well as cuboidal epithelial cells and macrophages. In contrast, PGHS-1 mRNA did not increase following ischemia. We conclude that PGHS-2 is present in piglet choroid plexus under normal conditions and that ischemia increases levels of PGHS-2 in choroid plexus.