Genes differentially expressed between pathogenic and non-pathogenic Entamoeba histolytica clones influence pathogenicity-associated phenotypes by multiple mechanisms.

Recently, two genes involved in amoebic liver abscess formation in a mouse model were identified by their differential expression of non-pathogenic (A1np) and pathogenic (B2p) clones of the Entamoeba histolytica isolate HM:1-IMSS. While overexpression of a gene encoding the metallopeptidase EhMP8-2 reduces the virulence of the pathogenic clone B2p, overexpression of the gene ehi_127670 (ehhp127), encoding a hypothetical protein, increases the virulence of the non-pathogenic clone A1np, while silencing this gene in the pathogenic B2p reduces virulence. To understand the role of both molecules in determining the pathogenicity of E. histolytica, silencing, and overexpression transfectants were characterized in detail. Silencing of ehmp8-2, of the homologous gene ehmp8-1, or both in non-pathogenic A1np trophozoites significantly altered the transcript levels of 347, 216, and 58 genes, respectively. This strong change in the expression profiles caused by the silencing of ehmp8-1 and ehmp8-2 implies that these peptidases regulate the expression of numerous genes. Consequently, numerous phenotypic characteristics, including cytopathic, hemolytic, and cysteine peptidase activity, were altered in response to their silencing. Silencing of ehhp127 in pathogenic B2p trophozoites did not affect the expression of other genes, whereas its overexpression in non-pathogenic A1np trophozoites results in an altered expression of approximately 140 genes. EhHP127 is important for trophozoite motility, as its silencing reduces, while its overexpression enhances movement activity. Interestingly, the specific silencing of ehhp127 also significantly affects cytopathic, cysteine peptidase, and hemolytic activities. All three molecules characterized in this study, namely EhMP8-1, EhMP8-2, and EhHP127, are present in amoeba vesicles. The results show that ehmp8-2 and ehhp127 are not only differentially expressed between pathogenic and non-pathogenic amoebae, but that they also significantly affect amoeba pathogenicity-associated phenotypes by completely different mechanisms. This observation suggests that the regulation of amoeba pathogenicity is achieved by a complex network of molecular mechanisms rather than by single factors.

Prognostic value of the micronucleus assay for clinical endpoints in neoadjuvant radiochemotherapy for rectal cancer.

BACKGROUND: The question whether lymphocyte radiosensitivity is representative of patients' response to radiotherapy (RT) remains unsolved. We analyzed lymphocyte cytogenetic damage in patients who were homogeneously treated with preoperative radiochemotherapy (RCT) for rectal cancer within clinical trials. We tested for interindividual variation and consistent radiosensitivity after in-vivo and in-vitro irradiation, analyzed the effect of patients' and RCT characteristics on cytogenetic damage, and tested for correlations with patients' outcome in terms of tumor response, survival and treatment-related toxicity. METHODS: The cytokinesis-block micronucleus cytome (CBMNcyt) assay was performed on the peripheral blood lymphocytes (PBLCs) of 134 patients obtained before, during, at the end of RCT, and during the 2-year follow-up. A subset of PBLCs obtained before RCT was irradiated in-vitro with 3 Gy. RCT included 50.4 Gy of pelvic RT with 5-fluorouracil (5-FU) alone (n = 78) or 5-FU plus oxaliplatin (n = 56). The analyzed variables included patients' age, gender, RT characteristics (planning target volume size [PTV size], RT technique), and chemotherapy characteristics (5-FU plasma levels, addition of oxaliplatin). Outcome was analyzed as tumor regression, patient survival, and acute and late toxicity. RESULTS: Cytogenetic damage increased significantly with the radiation dose and varied substantially between individuals. Women were more sensitive than men; no significant age-dependent differences were observed. There was a significant correlation between the cytogenetic damage after in-vitro irradiation and in-vivo RCT. We found a significant effect of the PTV size on the yields of cytogenetic damage after RCT, while the RT technique had no effect. Neither the addition of oxaliplatin nor the 5-FU levels influenced cytogenetic damage. We found no correlation between patient outcome and the cytogenetic damage. CONCLUSIONS: We found consistent cytogenetic damage in lymphocytes after in-vivo RCT and in-vitro irradiation. Gender was confirmed as a well-known, and the PTV size was identified as a less well-known influencing variable on lymphocyte cytogenetic damage after partial-body irradiation. A consistent level of cytogenetic damage after in-vivo and in-vitro irradiation may indicate the importance of genetic factors for individual radiosensitivity. However, we found no evidence that in-vivo or in-vitro irradiation-induced cytogenetic damage is an adequate biomarker for the response to RCT in rectal cancer patients.

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12) derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

Neuronal Vacuolization in Feline Panleukopenia Virus Infection.

Feline panleukopenia virus (FPV) infections are typically associated with anorexia, vomiting, diarrhea, neutropenia, and lymphopenia. In cases of late prenatal or early neonatal infections, cerebellar hypoplasia is reported in kittens. In addition, single cases of encephalitis are described. FPV replication was recently identified in neurons, although it is mainly found in cells with high mitotic activity. A female cat, 2 months old, was submitted to necropsy after it died with neurologic deficits. Besides typical FPV intestinal tract changes, multifocal, randomly distributed intracytoplasmic vacuoles within neurons of the thoracic spinal cord were found histologically. Next-generation sequencing identified FPV-specific sequences within the central nervous system. FPV antigen was detected within central nervous system cells, including the vacuolated neurons, via immunohistochemistry. In situ hybridization confirmed the presence of FPV DNA within the vacuolated neurons. Thus, FPV should be considered a cause for neuronal vacuolization in cats presenting with ataxia.

New Avian Hepadnavirus in Palaeognathous Bird, Germany.

In 2015, we identified an avian hepatitis B virus associated with hepatitis in a group of captive elegant-crested tinamous (Eudromia elegans) in Germany. The full-length genome of this virus shares <76% sequence identity with other avihepadnaviruses. The virus may therefore be considered a new extant avian hepadnavirus.

Differentiation of Staphylococcus argenteus (formerly: Staphylococcus aureus clonal complex 75) by mass spectrometry from S. aureus using the first strain isolated from a wild African great ape.

The species Staphylococcus argenteus was separated recently from Staphylococcus aureus (Tong S.Y., F. Schaumburg, M.J. Ellington, J. Corander, B. Pichon, F. Leendertz, S.D. Bentley, J. Parkhill, D.C. Holt, G. Peters, and P.M. Giffard, 2015). The objective of this work was to characterise the genome of a non-human S. argenteus strain, which had been isolated from the faeces of a wild-living western lowland gorilla in Gabon, and analyse the spectrum of this species in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The full genome sequence revealed a scarcity of virulence genes and absence of resistance genes, indicating a decreased virulence potential compared to S. aureus and the human methicillin-resistant S. argenteus isolate MSHR1132T. Spectra obtained by MALDI-TOF MS and the analysis of available sequences in the genome databases identified several MALDI-TOF MS signals that clearly differentiate S. argenteus, the closely related Staphylococcus schweitzeri and S. aureus. In conclusion, in the absence of biochemical tests that identify the three species, mass spectrometry should be employed as method of choice.

Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum.

Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

High-grade acute organ toxicity and p16(INK4A) expression as positive prognostic factors in primary radio(chemo)therapy for patients with head and neck squamous cell carcinoma.

BACKGROUND: Superior treatment response and survival for patients with human papilloma virus (HPV)-positive head and neck cancer (HNSCC) are documented in clinical studies. However, the relevance of high-grade acute organ toxicity (HGAOT), which has also been correlated with improved prognosis, has attracted scant attention in HPV-positive HNSCC patients. Hence we tested the hypothesis that both parameters, HPV and HGAOT, are positive prognostic factors in patients with HNSCC treated with definite radiotherapy (RT) or radiochemotherapy (RCT). PATIENTS AND METHODS: Pretreatment tumor tissue and clinical records were available from 233 patients receiving definite RT (62 patients) or RCT (171 patients). HPV infection was analysed by means of HPV DNA detection or p16(INK4A) expression; HGAOT was defined as the occurrence of acute organ toxicity >grade 2 according to the Common Toxicity Criteria. Both variables were correlated with overall survival (OS) using Cox proportional hazards regression. RESULTS: Positivity for HPV DNA (44 samples, 18.9 %) and p16(INK4A) expression (102 samples, 43.8 %) were significantly correlated (p < 0.01), and HGAOT occurred in 77 (33 %) patients. Overall, the 5-year OS was 23 %; stratified for p16(INK4A) expression and HGAOT, OS rates were 47 %, 42 %, 20 % and 10 % for patients with p16(INK4A) expression and HGAOT, patients with HGAOT only, patients with p16(INK4A) expression only, and patients without p16(INK4A) expression or HGAOT, respectively. After multivariate testing p16(INK4A) expression (p = 0.003) and HGAOT (p < 0.001) were significantly associated with OS. CONCLUSION: P16(INK4A) expression and HGAOT are independent prognostic factors for OS of patients with HNSCC, whereas p16(INK4A) expression is particularly important for patients without HGAOT.

Co-circulation of a novel phlebovirus and Massilia virus in sandflies, Portugal.

BACKGROUND: In Portugal, entomological surveys to detect phleboviruses in their natural vectors have not been performed so far. Thus, the aims of the present study were to detect, isolate and characterize phleboviruses in sandfly populations of Portugal. FINDINGS: From May to October 2007-2008, 896 female sandflies were trapped in Arrábida region, located on the southwest coast of Portugal. Phlebovirus RNA was detected by using a pan-phlebovirus RT-PCR in 4 out of 34 Phlebotomus perniciosus pools. Direct sequencing of the amplicons showed that 2 samples exhibited 72 % nucleotide identity with Arbia virus, and two showed 96 % nucleotide identity with Massilia virus. The Arbia-like virus (named Alcube virus) was isolated in cell culture and complete genomic sequences of one Alcube and two Massila viruses were determined using next-generation sequencing technology. Phylogenetic analysis demonstrated that Alcube virus clustered with members of the Salehabad virus species complex. Within this clade, Alcube virus forms a monophyletic lineage with the Arbia, Salehabad and Adana viruses sharing a common ancestor. Arbia virus has been identified as the most closely related virus with 20-28 % nucleotide and 10-27 % amino acid divergences depending on the analysed segment. CONCLUSIONS: We have provided genetic evidence for the circulation of a novel phlebovirus species named Alcube virus in Ph. perniciosus and co-circulation of Massilia virus, in Arrábida region, southwest of Portugal. Further epidemiological investigations and surveillance for sandfly-borne phleboviruses in Portugal are needed to elucidate their medical importance.

Assessing the utility of deep neural networks in detecting superficial surgical site infections from free text electronic health record data.

Background: High-quality outcomes data is crucial for continued surgical quality improvement. Outcomes are generally captured through structured administrative data or through manual curation of unstructured electronic health record (EHR) data. The aim of this study was to apply natural language processing (NLP) to chart notes in the EHR to accurately capture postoperative superficial surgical site infections (SSSIs). Methods: Deep Learning (DL) NLP models were trained on data from 389,865 surgical cases across all 11 hospitals in the Capital Region of Denmark. Surgical cases in the training dataset were performed between January 01st, 2017, and October 30th, 2021. We trained a forward reading and a backward reading universal language model on unlabeled postoperative chart notes recorded within 30 days of a surgical procedure. The two language models were subsequently finetuned on labeled data for the classification of SSSIs. Validation and testing were performed on surgical cases performed during the month of November 2021. We propose two different use cases: a stand-alone machine learning (SAM) pipeline and a human-in-the-loop (HITL) pipeline. Performances of both pipelines were compared to administrative data and to manual curation. Results: The models were trained on 3,983,864 unlabeled chart notes and finetuned on 1,231,656 labeled notes. Models had a test area under the receiver operating characteristic curves (ROC AUC) of 0.989 on individual chart notes and 0.980 on an aggregated case level. The SAM pipeline had a sensitivity of 0.604, a specificity of 0.996, a positive predictive value (PPV) of 0.763, and a negative predictive value (NPV) of 0.991. Prior to human review, the HITL pipeline had a sensitivity of 0.854, a specificity of 0.987, a PPV of 0.603, and a NPV of 0.997. Conclusion: The performance of the SAM pipeline was superior to administrative data, and significantly outperformed previously published results. The performance of the HITL pipeline approached that of manual curation.

Regulation of mammalian cell cycle progression in the regenerating liver.

The process of cell division in mammalian cells is orchestrated by cell-cycle-dependent oscillations of cyclin protein levels. Cyclin levels are controlled by redundant transcriptional, post-translational and degradation feedback loops. How each of these separate loops contributes to the regulation of the key cell cycle events and to the connection between the G1-S transition and the subsequent mitotic events is under investigation. Here, we present an integrated computational model of the mammalian cell cycle based on the sequential activation of cyclins. We validate the model against experimental data on liver cells (hepatocytes), which undergo one or two rounds of synchronous circadian-clock gated cell divisions during liver regeneration, after partial hepatectomy (PH). The model exhibits bandpass filter properties that allow the system to ignore strong but transient, or sustained but weak damages after PH. Bifurcation analysis of the model suggests two different threshold mechanisms for the progression of the cell through mitosis. These results are coherent with the notion that the mitotic exit in mammalian cells is bistable, and suggests that Cdc20 homologue 1 (Cdh1) is an important regulator of mitosis. Regulation by Cdh1 also explains the observed G2/M phase prolongation after hepatocyte growth factor (HGF) stimulation during S phase.

RHYTHM--a server to predict the orientation of transmembrane helices in channels and membrane-coils.

RHYTHM is a web server that predicts buried versus exposed residues of helical membrane proteins. Starting from a given protein sequence, secondary and tertiary structure information is calculated by RHYTHM within only a few seconds. The prediction applies structural information from a growing data base of precalculated packing files and evolutionary information from sequence patterns conserved in a representative dataset of membrane proteins ('Pfam-domains'). The program uses two types of position specific matrices to account for the different geometries of packing in channels and transporters ('channels') or other membrane proteins ('membrane-coils'). The output provides information on the secondary structure and topology of the protein and specifically on the contact type of each residue and its conservation. This information can be downloaded as a graphical file for illustration, a text file for analysis and statistics and a PyMOL file for modeling purposes. The server can be freely accessed at: URL: http://proteinformatics.de/rhythm.

Life Cycle Stage-Specific Accessibility of Leishmania donovani Chromatin at Transcription Start Regions.

Leishmania donovani is a parasitic protist that causes the lethal Kala-azar fever in India and East Africa. Gene expression in Leishmania is regulated by gene copy number variation and inducible translation while RNA synthesis initiates at a small number of sites per chromosome and proceeds through polycistronic transcription units, precluding a gene-specific regulation (C. Clayton and M. Shapira, Mol Biochem Parasitol 156:93-101, 2007, https://doi.org/10.1016/j.molbiopara.2007.07.007). Here, we analyze the dynamics of chromatin structure in both life cycle stages of the parasite and find evidence for an additional, epigenetic gene regulation pathway in this early branching eukaryote. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis (J. D. Buenrostro, P. G. Giresi, L. C. Zaba, H. Y. Chang, and W. J. Greenleaf, Nat Methods 10:1213-1218, 2013, https://doi.org/10.1038/nmeth.2688) predominantly shows euchromatin at transcription start regions in fast-growing promastigotes, but mostly heterochromatin in the slowly proliferating amastigotes, the mammalian stage, reflecting a previously shown increase of histone synthesis in the latter stage. IMPORTANCE Leishmania parasites are important pathogens with a global impact and cause poverty-related illness and death. They are devoid of classic cis- and trans-acting transcription regulators but use regulated translation and gene copy number variations to adapt to hosts and environments. In this work, we show that transcription start regions present as open euchromatin in fast-growing insect stages but as less-accessible heterochromatin in the slowly proliferating amastigote stage, indicating an epigenetic control of gene accessibility in this early branching eukaryotic pathogen. This finding should stimulate renewed interest in the control of RNA synthesis in Leishmania and related parasites.

Analysis of the Interaction Between Plasmodium falciparum-Infected Erythrocytes and Human Endothelial Cells Using a Laminar Flow System, Bioinformatic Tracking and Transcriptome Analysis.

During malaria infection, the endothelial lining of the small blood vessels of the brain and other vital organs is strongly stimulated. This leads to fatal complications and poor prognosis of the infection. It is believed that two main reasons are responsible for this pathology, namely the cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) on the one hand and the proinflammatory products released by the IEs which activate the endothelial cells (ECs) on the other hand. Until recently, most of the studies that characterized the activation of ECs were performed under static conditions, which do not reflect the real sequelae in vivo. In this chapter, we present a system, which allows authentic simulation of the IEs-ECs interactions during P. falciparum infection.The main idea of the system is to provide an adequate shear stress over the ECs during the cytoadhesion and stimulation with IEs, which provides a better basis for the investigation of the cytoadhesion pathology through analyzing the ECs' transcriptome after stimulation. On the other hand, analyzing the transcriptome of the IEs might also give deeper analysis of their response to shear stress. Deep understanding of these events might help in the development of novel treatment strategies that interfere with this cell-cell interaction.

Searching Novel Clock Genes Using RNAi-Based Screening.

RNA interference (RNAi) allows for the selective downregulation of gene expression by neutralizing targeted mRNA molecules and has frequently been used in high-throughput screening endeavors. Here, we describe a protocol for the highly parallel RNAi-mediated downregulation of gene expression in order to search for components involved in circadian rhythm generation. We use lentiviral gene transfer to deliver shRNA expressing plasmids into circadian reporter cells ensuring for efficient and stable knockdown. Circadian rhythms are monitored using live-cell bioluminescence recording of synchronized reporter cells over several days. In addition, we present a new software tool (ChronoStar) for efficient, parallel time-series analysis to extract rhythm parameters such as period, phase, amplitude, and damping.

Using machine learning for predicting intensive care unit resource use during the COVID-19 pandemic in Denmark.

The COVID-19 pandemic has put massive strains on hospitals, and tools to guide hospital planners in resource allocation during the ebbs and flows of the pandemic are urgently needed. We investigate whether machine learning (ML) can be used for predictions of intensive care requirements a fixed number of days into the future. Retrospective design where health Records from 42,526 SARS-CoV-2 positive patients in Denmark was extracted. Random Forest (RF) models were trained to predict risk of ICU admission and use of mechanical ventilation after n days (n = 1, 2, …, 15). An extended analysis was provided for n = 5 and n = 10. Models predicted n-day risk of ICU admission with an area under the receiver operator characteristic curve (ROC-AUC) between 0.981 and 0.995, and n-day risk of use of ventilation with an ROC-AUC between 0.982 and 0.997. The corresponding n-day forecasting models predicted the needed ICU capacity with a coefficient of determination (R2) between 0.334 and 0.989 and use of ventilation with an R2 between 0.446 and 0.973. The forecasting models performed worst, when forecasting many days into the future (for large n). For n = 5, ICU capacity was predicted with ROC-AUC 0.990 and R2 0.928, and use of ventilator was predicted with ROC-AUC 0.994 and R2 0.854. Random Forest-based modelling can be used for accurate n-day forecasting predictions of ICU resource requirements, when n is not too large.

Altered Cytokine Response of Human Brain Endothelial Cells after Stimulation with Malaria Patient Plasma.

Infections with the deadliest malaria parasite, Plasmodium falciparum, are accompanied by a strong immunological response of the human host. To date, more than 30 cytokines have been detected in elevated levels in plasma of malaria patients compared to healthy controls. Endothelial cells (ECs) are a potential source of these cytokines, but so far it is not known if their cytokine secretion depends on the direct contact of the P. falciparum-infected erythrocytes (IEs) with ECs in terms of cytoadhesion. Culturing ECs with plasma from malaria patients (27 returning travellers) resulted in significantly increased secretion of IL-11, CXCL5, CXCL8, CXCL10, vascular endothelial growth factor (VEGF) and angiopoietin-like protein 4 (ANGPTL4) if compared to matching controls (22 healthy individuals). The accompanying transcriptome study of the ECs identified 43 genes that were significantly increased in expression (≥1.7 fold) after co-incubation with malaria patient plasma, including cxcl5 and angptl4. Further bioinformatic analyses revealed that biological processes such as cell migration, cell proliferation and tube development were particularly affected in these ECs. It can thus be postulated that not only the cytoadhesion of IEs, but also molecules in the plasma of malaria patients exerts an influence on ECs, and that not only the immunological response but also other processes, such as angiogenesis, are altered.

Developing and validating COVID-19 adverse outcome risk prediction models from a bi-national European cohort of 5594 patients.

Patients with severe COVID-19 have overwhelmed healthcare systems worldwide. We hypothesized that machine learning (ML) models could be used to predict risks at different stages of management and thereby provide insights into drivers and prognostic markers of disease progression and death. From a cohort of approx. 2.6 million citizens in Denmark, SARS-CoV-2 PCR tests were performed on subjects suspected for COVID-19 disease; 3944 cases had at least one positive test and were subjected to further analysis. SARS-CoV-2 positive cases from the United Kingdom Biobank was used for external validation. The ML models predicted the risk of death (Receiver Operation Characteristics-Area Under the Curve, ROC-AUC) of 0.906 at diagnosis, 0.818, at hospital admission and 0.721 at Intensive Care Unit (ICU) admission. Similar metrics were achieved for predicted risks of hospital and ICU admission and use of mechanical ventilation. Common risk factors, included age, body mass index and hypertension, although the top risk features shifted towards markers of shock and organ dysfunction in ICU patients. The external validation indicated fair predictive performance for mortality prediction, but suboptimal performance for predicting ICU admission. ML may be used to identify drivers of progression to more severe disease and for prognostication patients in patients with COVID-19. We provide access to an online risk calculator based on these findings.

Predicting protein complex geometries with a neural network.

A major challenge of the protein docking problem is to define scoring functions that can distinguish near-native protein complex geometries from a large number of non-native geometries (decoys) generated with noncomplexed protein structures (unbound docking). In this study, we have constructed a neural network that employs the information from atom-pair distance distributions of a large number of decoys to predict protein complex geometries. We found that docking prediction can be significantly improved using two different types of polar hydrogen atoms. To train the neural network, 2000 near-native decoys of even distance distribution were used for each of the 185 considered protein complexes. The neural network normalizes the information from different protein complexes using an additional protein complex identity input neuron for each complex. The parameters of the neural network were determined such that they mimic a scoring funnel in the neighborhood of the native complex structure. The neural network approach avoids the reference state problem, which occurs in deriving knowledge-based energy functions for scoring. We show that a distance-dependent atom pair potential performs much better than a simple atom-pair contact potential. We have compared the performance of our scoring function with other empirical and knowledge-based scoring functions such as ZDOCK 3.0, ZRANK, ITScore-PP, EMPIRE, and RosettaDock. In spite of the simplicity of the method and its functional form, our neural network-based scoring function achieves a reasonable performance in rigid-body unbound docking of proteins. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Rapid dark repression of 5-aminolevulinic acid synthesis in green barley leaves.

In photosynthetic organisms chlorophyll and heme biosynthesis is tightly regulated at various levels in response to environmental adaptation and plant development. The formation of 5-aminolevulinic acid (ALA) is the key regulatory step and provides adequate amounts of the common precursor molecule for the Mg and Fe branches of tetrapyrrole biosynthesis. Pathway control prevents accumulation of metabolic intermediates and avoids photo-oxidative damage. In angiosperms reduction of protochlorophyllide (Pchlide) to chlorophyllide is catalyzed by the light-dependent NADPH:Pchlide oxidoreductase (POR). Although a correlation between down-regulated ALA synthesis and accumulation of Pchlide in the dark was proposed a long time ago, the time-resolved mutual dependency has never been analyzed. Taking advantage of the high metabolic activity of young barley (Hordeum vulgare L.) seedlings, in planta ALA synthesis could be determined with high time-resolution. ALA formation declined immediately after transition from light to dark and correlated with an immediate accumulation of POR-bound Pchlide within the first 60 min in darkness. The flu homologous barley mutant tigrina d(12) uncouples ALA synthesis from dark-suppression and continued to form ALA in darkness without a significant change in synthesis rate in this time interval. Similarly, inhibition of protoporphyrinogen IX oxidase by acifluorfen resulted in a delayed accumulation of Pchlide during the entire dark period and a weak repression of ALA synthesis in darkness. Moreover, it is demonstrated that dark repression of ALA formation relies rather on rapid post-translational regulation in response to accumulating Pchlide than on changes in nuclear gene expression.