A Texture-based land cover classification for the delineation of a shifting cultivation landscape in the Lao PDR using landscape metrics

The delineation of shifting cultivation landscapes using remote sensing in mountainous regions is challenging. On the one hand, there are difficulties related to the distinction of forest and fallow forest classes as occurring in a shifting cultivation landscape in mountainous regions. On the other hand, the dynamic nature of the shifting cultivation system poses problems to the delineation of landscapes where shifting cultivation occurs. We present a two-step approach based on an object-oriented classification of Advanced Land Observing Satellite, Advanced Visible and Near-Infrared Spectrometer (ALOS AVNIR) and Panchromatic Remote-sensing Instrument for Stereo Mapping (ALOS PRISM) data and landscape metrics. When including texture measures in the object-oriented classification, the accuracy of forest and fallow forest classes could be increased substantially. Based on such a classification, landscape metrics in the form of land cover class ratios enabled the identification of crop-fallow rotation characteristics of the shifting cultivation land use practice. By classifying and combining these landscape metrics, shifting cultivation landscapes could be delineated using a single land cover dataset.

Bifurcation and singularity analysis of a molecular network for the induction of long-term memory.

Withdrawal reflexes of the mollusk Aplysia exhibit sensitization, a simple form of long-term memory (LTM). Sensitization is due, in part, to long-term facilitation (LTF) of sensorimotor neuron synapses. LTF is induced by the modulatory actions of serotonin (5-HT). Pettigrew et al. developed a computational model of the nonlinear intracellular signaling and gene network that underlies the induction of 5-HT-induced LTF. The model simulated empirical observations that repeated applications of 5-HT induce persistent activation of protein kinase A (PKA) and that this persistent activation requires a suprathreshold exposure of 5-HT. This study extends the analysis of the Pettigrew model by applying bifurcation analysis, singularity theory, and numerical simulation. Using singularity theory, classification diagrams of parameter space were constructed, identifying regions with qualitatively different steady-state behaviors. The graphical representation of these regions illustrates the robustness of these regions to changes in model parameters. Because persistent protein kinase A (PKA) activity correlates with Aplysia LTM, the analysis focuses on a positive feedback loop in the model that tends to maintain PKA activity. In this loop, PKA phosphorylates a transcription factor (TF-1), thereby increasing the expression of an ubiquitin hydrolase (Ap-Uch). Ap-Uch then acts to increase PKA activity, closing the loop. This positive feedback loop manifests multiple, coexisting steady states, or multiplicity, which provides a mechanism for a bistable switch in PKA activity. After the removal of 5-HT, the PKA activity either returns to its basal level (reversible switch) or remains at a high level (irreversible switch). Such an irreversible switch might be a mechanism that contributes to the persistence of LTM. The classification diagrams also identify parameters and processes that might be manipulated, perhaps pharmacologically, to enhance the induction of memory. Rational drug design, to affect complex processes such as memory formation, can benefit from this type of analysis.

A model of late long-term potentiation simulates aspects of memory maintenance.

Late long-term potentiation (L-LTP) denotes long-lasting strengthening of synapses between neurons. L-LTP appears essential for the formation of long-term memory, with memories at least partly encoded by patterns of strengthened synapses. How memories are preserved for months or years, despite molecular turnover, is not well understood. Ongoing recurrent neuronal activity, during memory recall or during sleep, has been hypothesized to preferentially potentiate strong synapses, preserving memories. This hypothesis has not been evaluated in the context of a mathematical model representing ongoing activity and biochemical pathways important for L-LTP. In this study, ongoing activity was incorporated into two such models - a reduced model that represents some of the essential biochemical processes, and a more detailed published model. The reduced model represents synaptic tagging and gene induction simply and intuitively, and the detailed model adds activation of essential kinases by Ca(2+). Ongoing activity was modeled as continual brief elevations of Ca(2+). In each model, two stable states of synaptic strength/weight resulted. Positive feedback between synaptic weight and the amplitude of ongoing Ca(2+) transients underlies this bistability. A tetanic or theta-burst stimulus switches a model synapse from a low basal weight to a high weight that is stabilized by ongoing activity. Bistability was robust to parameter variations in both models. Simulations illustrated that prolonged periods of decreased activity reset synaptic strengths to low values, suggesting a plausible forgetting mechanism. However, episodic activity with shorter inactive intervals maintained strong synapses. Both models support experimental predictions. Tests of these predictions are expected to further understanding of how neuronal activity is coupled to maintenance of synaptic strength. Further investigations that examine the dynamics of activity and synaptic maintenance can be expected to help in understanding how memories are preserved for up to a lifetime in animals including humans.

Tuning Molecular Recognition of RNA and DNA via Sugar Modification

This minireview highlights three aspects of our recent work in the area of sugar modified oligonucleotide analogues. It provides an overview over recent results on the conformationally constrained analogue tricyclo-DNA with special emphasis of its antisense properties, it summarizes results on triple-helix forming oligodeoxynucleotides containing pyrrolidino-nucleosides with respect to DNA recognition via the dual recognition mode, and it highlights the advantageous application of the orthogonal oligonucleotidic pairing system homo-DNA in molecular beacons for DNA diagnostics

Tissue metabolomics of hepatocellular carcinoma: Tumor energy metabolism and the role of transcriptomic classification

Hepatocellular carcinoma (HCC) is one of the commonest causes of death from cancer. A plethora of metabolomic investigations of HCC have yielded molecules in biofluids that are both up- and down-regulated but no real consensus has emerged regarding exploitable biomarkers for early detection of HCC. We report here a different approach, a combined transcriptomics and metabolomics study of energy metabolism in HCC. A panel of 31 pairs of HCC tumors and corresponding nontumor liver tissues from the same patients was investigated by gas chromatography-mass spectrometry (GCMS)-based metabolomics. HCC was characterized by ∼2-fold depletion of glucose, glycerol 3- and 2-phosphate, malate, alanine, myo-inositol, and linoleic acid. Data are consistent with a metabolic remodeling involving a 4-fold increase in glycolysis over mitochondrial oxidative phosphorylation. A second panel of 59 HCC that had been typed by transcriptomics and classified in G1 to G6 subgroups was also subjected to GCMS tissue metabolomics. No differences in glucose, lactate, alanine, glycerol 3-phosphate, malate, myo-inositol, or stearic acid tissue concentrations were found, suggesting that the Wnt/β-catenin pathway activated by CTNNB1 mutation in subgroups G5 and G6 did not exhibit specific metabolic remodeling. However, subgroup G1 had markedly reduced tissue concentrations of 1-stearoylglycerol, 1-palmitoylglycerol, and palmitic acid, suggesting that the high serum α-fetoprotein phenotype of G1, associated with the known overexpression of lipid catabolic enzymes, could be detected through metabolomics as increased lipid catabolism. Conclusion: Tissue metabolomics yielded precise biochemical information regarding HCC tumor metabolic remodeling from mitochondrial oxidation to aerobic glycolysis and the impact of molecular subtypes on this process.

Bacteriological diagnosis and molecular strain typing of Mycobacterium bovis and Mycobacterium caprae

The primary isolation of a Mycobacterium sp. of the Mycobacterium tuberculosis complex from an infected animal provides a definitive diagnosis of tuberculosis. However, as Mycobacterium bovis and Mycobacterium caprae are difficult to isolate, particularly for animals in the early stages of disease, success is dependent on the optimal performance of all aspects of the bacteriological process, from the initial choice of tissue samples at post-mortem examination or clinical samples, to the type of media and conditions used to cultivate the microorganism. Each step has its own performance characteristics, which can contribute to sensitivity and specificity of the procedure, and may need to be optimized in order to achieve the gold standard diagnosis. Having isolated the slow-growing mycobacteria, species identification and fine resolution strain typing are keys to understanding the epidemiology of the disease and to devise strategies to limit transmission of infection. New technologies have emerged that can now even discriminate different isolates from the same animal. In this review we highlight the key factors that contribute to the accuracy of bacteriological diagnosis of M. bovis and M. caprae, and describe the development of advanced genotyping techniques that are increasingly used in diagnostic laboratories for the purpose of supporting detailed epidemiological investigations.

Towards the introduction of the 'Immunoscore' in the classification of malignant tumours.

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Morphological and molecular characteristics of HER2 amplified urothelial bladder cancer.

Several (pre-) clinical trials are currently investigating the benefit of HER2-targeted therapy in urothelial bladder cancer (UBC). Patients with HER2 amplified UBC could potentially profit from these therapies. However, little is known about histomorphology, HER2 protein expression patterns and occurrence of alterations in the HER2 gene in their tumors. Among 150 metastasizing primary UBC, 13 HER2 amplified tumors were identified. Their histopathological features were compared with 13 matched, non-amplified UBC. HER2 protein expression was determined by immunohistochemistry. The 26 tumors were screened for mutations in exons 19 and 20 of the HER2 gene. UBC with HER2 amplification presented with a broad variety of histological variants (median 2 vs. 1), frequently featured micropapillary tumor components (77 % vs. 8 %) and demonstrated a high amount of tumor associated inflammation. Immunohistochemically, 10 of 13 (77 %) HER2 amplified tumors were strongly HER2 protein positive. Three tumors (23 %) were scored as HER2 negative. One of the HER2 amplified tumors harbored a D769N mutation in exon 19 of the HER2 gene; all other tested tumors were wild type. In conclusion, HER2 amplified UBC feature specific morphological characteristics. They frequently express the HER2 protein diffusely and are, therefore, promising candidates for HER2 targeted therapies. The detection of mutations at the HER2 locus might add new aspects to molecular testing of UBC.

Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations

We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution to investigate interaction and binding of α-tochoperol transfer protein (α-TTP) to phosphatidylinositol phosphate lipids (PIPs). Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP-PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein/ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED) phenotypes. Specifically, R221 is main residue responsible for the stabilization of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP.

Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death.

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called "damage-associated molecular patterns" (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.

In vivo definition of genetic and cellular aspects of mammalian sexual differentiation

The differentiation of the reproductive organs is an essential developmental process required for the proper transmission of the genetic material. Müllerian inhibiting substance (MIS) is produced by testes and is necessary for the regression of the Müllerian ducts: the anlagen of the uterus, fallopian tubes and cervix. In vitro and standard transgenic mouse studies indicate that the nuclear hormone receptor Steroidogenic factor 1 (SF-1) and the transcription factor SOX9 play an essential role in the regulation of Mis. To test this hypothesis, mutations in the endogenous SF-1 and SOX9 binding sites in the mouse Mis promoter were introduced by gene targeting in embryonic stem (ES) cells. In disagreement with cell culture and transgenic mouse studies, male mice homozygous for the mutant SF-1 binding site correctly initiated Mis transcription in the fetal testes, although at significantly reduced levels. Surprisingly, sufficient Mis was produced for complete elimination of the Müllerian duct system. However, when the SF-1 binding site mutation was combined with an Mis -null allele, the further decrease in Mis levels led to a partial retention of uterine tissue, but only at a distance from the testes. In contrast, males homozygous for the mutant SOX9 binding site did not initiate Mis transcription, resulting in pseudohermaphrodites with a uterus and oviducts. These studies suggest an essential role for SOX9 in the initiation of Mis transcription, whereas SF-1 appears to act as a quantitative regulator of Mis transcript levels perhaps for influencing non-Müllerian duct tissues. ^ The Mis type II receptor, a member of the TGF- b superfamily, is also required for the proper regression of the Müllerian ducts. Mis type II receptor-deficient human males and their murine counterparts develop as pseudohermaphrodites. A lacZ reporter cassette was introduced into the mouse Mis type II receptor gene, by homologous recombination in ES cells. Expression studies, based on b -galactosidase activity, show marked expression of the MIS type II receptor in the postnatal Sertoli cells of the testis as well as in the prenatal and postnatal granulosa cells of the ovary. Expression is also seen in the mesenchymal cells surrounding the Müllerian duct and in the longitudinal muscle layer of the uterus. ^

New aspects of estrogen action in the endometrium: Reciprocal interplay with retinoic acid pathway and a novel estrogen-regulated gene

The uterine endometrium is a major target for the estrogen. However, the molecular basis of estrogen action in the endometrium is largely unknown. I have used two approaches to study the effects of estrogen on the endometrium. One approach involved the study of the interaction between estrogen and retinoic acid (RA) pathways in the endometrium. I have demonstrated that estrogen administration to rodents and estrogen replacement therapy (ERT) in postmenopausal women selectively induced the endometrial expression of retinaldehyde dehydrogenase II (RALDH2), a critical enzyme of RA biosynthesis. RALDH2 was expressed exclusively in the stromal cells, especially in the stroma adjacent to the luminal and glandular epithelia. The induction of RALDH2 by estrogen required estrogen receptor and occurred via a direct increase in RALDH2 transcription. Among the three RA receptors, estrogen selectively induced the expression of RARα. In parallel, estrogen also increased the utilization of all-trans retinol (the substrate for RA biosynthesis) and the expression of two RA-regulated marker genes, cellular retinoic acid binding protein II (CRABP2) and tissue transglutaminase (tTG) in the endometrium. Thus estrogen coordinately upregulated both the production and signaling of RA in both the rodent and human endometrium. This coordinate upregulation of RA system appeared to play a role in counterbalancing the stimulatory effects of estrogen on the endometrium, since the depletion of endogenous RA in mice led to an increase in estrogen-stimulated stromal proliferation and endometrial Akt phosphorylation. In addition, I have also used a systematic approach (DNA microarray) to categorize genes and pathways affected by the ERT in the endometrium of postmenopausal women and identified a novel estrogen-regulated gene EIG121. EIG121 was exclusively expressed in the glandular epithelial cells of the endometrium and induced by estrogen in vivo and in cultured cell lines. Compared with the normal endometrium, EIG121 was highly overexpressed in type 1 endometrial cancer, but profoundly suppressed in type 2 endometrial tumors. Taken together, these studies suggested that estrogen regulates the expression of many genes of both the pro-proliferative and anti-proliferative pathways and the abnormality of these pathways may increase the risks for estrogen-dependent endometrial hyperplasia and endometrial cancer. ^

Identification and characterization of a molecular marker in epithelial ovarian cancer

Carcinomas that arise from the ovarian surface epithelium represent a great challenge in gynecologic oncology. Although the prognosis of ovarian cancer is influenced by many factors capable of predicting clinical outcome, including tumor stage, pathological grade, and amount of residual disease following primary surgery, the biological aspects of ovarian cancer are not completely understood, thus implying that there may be other predictive indicators that could be used independently or in conjunction with these factors to provide a clearer clinical picture. The identification of additional markers with biological relevance is desirable. To identify disease-associated peptides, a phage display random peptide library was used to screen immunoglobulins derived from a patient with ovarian cancer. One peptide was markedly enriched following three rounds of affinity selection. The presence of autoantibodies against the peptide was examined in a panel of ovarian cancer patients. Stage IV patients exhibited a high percentage of positive reactivity (59%). This was in contrast to stage III patients, who only displayed 7% positive reactivity. Antibodies against the peptide were affinity purified, and heat-shock protein 90 (Hsp90) was identified as the corresponding autoantigen. The expression profile of the identified antigen was determined. Hsp90 was expressed in all sections examined regardless of degree of anaplasia. This thesis shows that utilizing the humoral response to ovarian cancer can be used to identify a tumor antigen in ovarian cancer. The data show that certain antigens may be expressed in ovarian tumors independent of the disease stage or grade, whereas circulating antibodies against such epitopes are only found in a subset of patients. ^

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Spatial and temporal aspects of synaptic plasticity

The notion that changes in synaptic efficacy underlie learning and memory processes is now widely accepted even if definitive proof of the synaptic plasticity and memory hypothesis is still lacking. When learning occurs, patterns of neural activity representing the occurrence of events cause changes in the strength of synaptic connections within the brain. Reactivation of these altered connections constitutes the experience of memory for these events and for other events with which they may be associated. These statements summarize a long-standing theory of memory formation that we refer to as the synaptic plasticity and memory hypothesis. Since activity-dependent synaptic plasticity is induced at appropriate synapses during memory formation, and is both necessary and sufficient for the information storage, we can speculate that a methodological study of the synapse will help us understand the mechanism of learning. Random events underlie a wide range of biological processes as diverse as genetic drift and molecular diffusion, regulation of gene expression and neural network function. Additionally spatial variability may be important especially in systems with nonlinear behavior. Since synapse is a complex biological system we expect that stochasticity as well as spatial gradients of different enzymes may be significant for induction of plasticity. ^ In that study we address the question "how important spatial and temporal aspects of synaptic plasticity may be". We developed methods to justify our basic assumptions and examined the main sources of variability of calcium dynamics. Among them, a physiological method to estimate the number of postsynaptic receptors as well as a hybrid algorithm for simulating postsynaptic calcium dynamics. Additionally we studied how synaptic geometry may enhance any possible spatial gradient of calcium dynamics and how that spatial variability affect plasticity curves. Finally, we explored the potential of structural synaptic plasticity to provide a metaplasticity mechanism specific for the synapse. ^