Multi-Target Analysis and Design of Mitochondrial Metabolism

Analyzing and optimizing biological models is often identified as a research priority in biomedical engineering. An important feature of a model should be the ability to find the best condition in which an organism has to be grown in order to reach specific optimal output values chosen by the researcher. In this work, we take into account a mitochondrial model analyzed with flux-balance analysis. The optimal design and assessment of these models is achieved through single- and/or multi-objective optimization techniques driven by epsilon-dominance and identifiability analysis. Our optimization algorithm searches for the values of the flux rates that optimize multiple cellular functions simultaneously. The optimization of the fluxes of the metabolic network includes not only input fluxes, but also internal fluxes. A faster convergence process with robust candidate solutions is permitted by a relaxed Pareto dominance, regulating the granularity of the approximation of the desired Pareto front. We find that the maximum ATP production is linked to a total consumption of NADH, and reaching the maximum amount of NADH leads to an increasing request of NADH from the external environment. Furthermore, the identifiability analysis characterizes the type and the stage of three monogenic diseases. Finally, we propose a new methodology to extend any constraint-based model using protein abundances.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

Quantitation of Malaria Parasite-Erythrocyte Cell-Cell Interactions Using Optical Tweezers

Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria.     

SAR study and conformational analysis of a series of novel peptide G protein‐coupled receptor kinase 2 inhibitors

G protein‐coupled receptor kinase 2 (GRK2) plays a central role in the cellular transduction network. In particular, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. Thereby, its inhibition offers a potential therapeutic solution to several pathological conditions. In the present study, we performed a SAR study and a NMR conformational analysis of peptides derived from HJ loop of GRK2 and able to selectively inhibit GRK2. From Ala‐scan and d ‐Ala point replacement, we found that Arg residues don't affect the inhibitory properties, while a d ‐amino acid at position 5 is key to the activity. Conformational analysis identified two β‐turns that involve N‐terminal residues, followed by a short extended region. These information can help the design of peptides and peptido‐mimetics with enhanced GRK2 inhibition properties. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 121–128, 2014.     

Morphological and Spatio‐Temporal Mismatches Shape a Neotropical Savanna Plant‐Hummingbird Network

Complex networks of species interactions might be determined by species traits but also by simple chance meetings governed by species abundances. Although the idea that species traits structure mutualistic networks is appealing, most studies have found abundance to be a major structuring mechanism underlying interaction frequencies. With a well‐resolved plant–hummingbird interaction network from the Neotropical savanna in Brazil, we asked whether species morphology, phenology, nectar availability and habitat occupancy and/or abundance best predicted the frequency of interactions. For this, we constructed interaction probability matrices and compared them to the observed plant‐hummingbird matrix through a likelihood approach. Furthermore, a recently proposed modularity algorithm for weighted bipartite networks was employed to evaluate whether these factors also scale‐up to the formation of modules in the network. Interaction frequencies were best predicted by species morphology, phenology and habitat occupancy, while species abundances and nectar availability performed poorly. The plant–hummingbird network was modular, and modules were associated to morphological specialization and habitat occupancy. Our findings highlight the importance of traits as determinants of interaction frequencies and network structure, corroborating the results of a previous study on a plant–hummingbird network from the Brazilian Atlantic Forest. Thus, we propose that traits matter more in tropical plant–hummingbird networks than in less specialized systems. To test the generality of this hypothesis, future research could employ geographic or taxonomic cross‐system comparisons contrasting networks with known differences in level of specialization.     

Novel induced <Emphasis Type="Italic">mlo</Emphasis> mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein

Background  

Recessively inherited natural and induced mutations in the barley Mlo gene confer durable broad-spectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Mlo codes for a member of a plant-specific family of polytopic integral membrane proteins with unknown biochemical activity. Resistant barley mlo mutant alleles identify amino acid residues that are critical for Mlo function in the context of powdery mildew susceptibility.     

Identification of Targeted Analyte Clusters for Studies of Schizophrenia

The search for biomarkers to diagnose psychiatric disorders such as schizophrenia has been underway for decades. Many molecular profiling studies in this field have focused on identifying individual marker signals that show significant differences in expression between patients and the normal population. However, signals for multiple analyte combinations that exhibit patterned behaviors have been less exploited. Here, we present a novel approach for identifying biomarkers of schizophrenia using expression of serum analytes from first onset, drug-naïve patients and normal controls. The strength of patterned signals was amplified by analyzing data in reproducing kernel spaces. This resulted in the identification of small sets of analytes referred to as targeted clusters that have discriminative power specifically for schizophrenia in both human and rat models. These clusters were associated with specific molecular signaling pathways and less strongly related to other neuropsychiatric disorders such as major depressive disorder and bipolar disorder. These results shed new light concerning how complex neuropsychiatric diseases behave at the pathway level and demonstrate the power of this approach in identification of disease-specific biomarkers and potential novel therapeutic strategies.Schizophrenia is a debilitating neuropsychiatric disorder that affects more than 1% of the world population and costs hundreds of billions of United States dollars in healthcare provision and lost earnings (1). The diagnosis of this disease has not changed substantially over several decades and currently relies on subjective psychopathological ratings such as the Diagnostic and Statistical Manual of Mental Disorders (DSM)1-IV. Thus, diagnosis can be complicated by the presence of overlapping symptoms frequently occurring in other psychiatric illnesses such as bipolar disorder (BD) and major depressive disorder (MDD) and by the presence of confounding factors such as drug abuse and co-morbidities. This often results in diagnosis being delayed for several months to years. A delay in establishing an accurate diagnosis can have serious deleterious implications because a late or imprecise diagnosis can contribute to unsatisfactory outcomes to currently used drug therapies and to higher rates of relapse (2). Most importantly, more than half of schizophrenia subjects develop a progressive course of the disease associated with deficit symptoms (3).In contrast, early therapeutic intervention holds promise in preventing or diminishing such effects (4–6). An empirical assay for early and accurate diagnosis of schizophrenia would deliver improved patient outcomes and reduce the costs of the disease for healthcare services and society (7–9). Such an assay could also provide a means of stratifying patients and monitoring drug responses and may also lead to the development of translational medicine tools that are critical for discovery of novel therapeutic strategies. Molecular profiling methods that afford the simultaneous measurement of multiple analytes in clinical and preclinical samples have considerable promise in this endeavor. These methods have been aimed predominantly at identifying individual molecules that show differences in expression between the disease and control conditions. However, such studies have often been fraught with small fold-changes in analyte levels, a common obstacle when investigating complex neuropsychiatric disorders (10–12). Thus, standard statistical techniques such as t tests will not be able to explore patterned behaviors involving proteins that have subtle expression changes but still contribute to the development of schizophrenia.The main objective of this study was to determine whether unique patterns of biomarkers can be identified for subjects with first onset antipsychotic-naïve schizophrenia. Analyte expression lists were generated using the Multi-Analyte Profiling (MAP®) fluorescent bead-based technology for profiling serum samples from 77 male schizophrenia patients and 66 matched male controls. For comparison with other psychiatric disorders, we also analyzed the serum samples of 13 male BD and 17 male MDD patients. In parallel, serum samples from four relevant animal models were also profiled for comparison with the human disease state. Analysis of the respective expression lists was carried out using non-linear statistical analysis, which identifies small sets of analytes called targeted analyte clusters (TACs) that have the power to discriminate the patients from normal controls. We present here the performance of these clusters for diagnosis of schizophrenia. In addition, we show how this method can also contribute to increasing our understanding of the etiology of the disorder by determining its ability to classify various preclinical models of psychiatric disorders. The biological pathways associated with these clusters are discussed with their relevance to schizophrenia.     

Nuclear Dynamics during Germination,Conidiation, and Hyphal Fusion of Fusarium oxysporum

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.Fusarium oxysporum is a soilborne pathogen that causes substantial losses in a wide variety of crops (12) and has been reported as an emerging human pathogen (36, 38). Similar to other fungal pathogens (18), the early stages of interaction between F. oxysporum and the host are crucial for the outcome of infection (11). Key processes occurring during these initial stages include spore germination, adhesion to the host surface, establishment of hyphal networks through vegetative hyphal fusion, differentiation of infection hyphae, and penetration of the host (53). Surprisingly, very little is known about the cytology of basic processes, such as spore germination and hyphal development, which play key roles during infection by F. oxysporum.F. oxysporum produces three types of asexual spores: microconidia, macroconidia, and chlamydospores (9, 26). Germination usually represents the first step in the colonization of a new environment, including the host. Once dormancy is broken, spores undergo a defined set of morphogenetic changes that lead to the establishment of a polarized growth axis and the emergence of one or multiple germ tubes (reviewed by d''Enfert and Hardham [10, 19]). In certain fungi, such as Aspergillus nidulans, germ tube emergence and septum formation are subject to precise spatial controls and are tightly coordinated with nuclear division (20, 22, 34, 42, 54). In contrast, in spores from other filamentous fungi, such as macroconidia of Fusarium graminearum, nuclear division is not required for the emergence of germ tubes (21, 48). During hyphal growth, multinucleate fungi display distinct mitotic patterns, such as asynchronous nuclear division in Neurospora crassa and Ashbya gossypii (15, 16, 29, 30, 33, 49), parasynchronous in A. nidulans (7, 15, 23, 46), and synchronous in Ceratocystis fagacearum (1, 15).Vegetative hyphal fusion, or anastomosis, is a common developmental process during the life cycle of filamentous fungi that is thought to serve important functions in intrahyphal communication, nutrient transport, and colony homeostasis (41). F. oxysporum undergoes anastomosis (8, 25, 32, 40), and although this process is not strictly required for plant infection, it appears to contribute to efficient colonization of the root surface (39).The aim of this study was to explore nuclear dynamics during different developmental stages of F. oxysporum that are of key relevance during the establishment of infection. They include germination of microconidia, vegetative hyphal development, and conidiation, as well as vegetative hyphal fusion during colony establishment. Fusion PCR-mediated gene targeting (55) was used to C-terminally label histone H1 in F. oxysporum (FoH1) with either green fluorescent protein (GFP) or the cherry variant (ChFP), allowing us to perform, for the first time, live-cell analysis of nuclear dynamics in this species. Our study revealed distinct patterns of nuclear divisions in F. oxysporum. Moreover, we report, for the first time in an ascomycete, that hyphal fusion initiates a series of nuclear events, including mitosis in the fusing hypha and nuclear migration into the receptor hypha, followed by degradation of the resident nucleus.