Evaluation of the rates of biological processes from tracer kinetic data. IV. Digital simulation of nucleic acid metabolism in bacteria

A flexible computer program was set up to simulate the kinetics of the amounts of radioactivity and the amounts of substance in various classes of nucleic acids for a generalized biological system. This program works by transferring numbers from registers to other registers corresponding to the amounts of isotope label or the amount of substance moving from one pool or compartment to another for each successive time interval. It is practical to choose the time interval small enough so that the same results are accurately obtained with this type of computation as with the analytical solution for the most realistic and complicated case previously formulated. Much more complicated situations now can be treated with this recursion program and questions of relevance to current molecular biology can be answered. The questions treated in this paper are directed to enteric microorganisms and are as follows. (1) What are the conditions under which a short pulse reflects net synthesis and what are the conditions where it reflects total synthesis? (2) What are the conditions that measurement of intermediate pool specific activity can yield information about the turnover and concentration of unstable macromolecules in the system? (3) How prolonged can a pulse be and still label the species of RNA substantially in proportion to their rates of synthesis? (4) What are the amounts and rates of synthesis and degradation of ribosomal precursors best fitting data in the literature?     

Fifteen compelling open questions in plant cell biology

As scientists, we are at least as excited about the open questions—the things we do not know—as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such “rules” conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.

We asked 15 experts to address what they consider to be the most compelling open questions in plant cell biology and these are their questions.     

The role of DNA fragmentation in apoptosis

The formation of distinct DNA fragments of oligonucleosomal size (180-200 bp lengths) is a biochemical hallmark of apoptosis in many cells. Recent observations also suggest large DNA fragments and even single-strand cleavage events occur during cell death. These observations have raised many questions. What are the types of DNA cleavage observed during apoptosis? What are the nucleases involved? And what is the role of these nucleolytic events in apoptosis?     

The genetics and molecular biology of the titin/connectin-like proteins of invertebrates

Conclusions A substantial amount of information has been gathered about the structure and function of twitchin/titin-related proteins in the invertebrates. This has been obtained through sequence analysis and the analysis of loss-offunction phenotypes inC. elegans andDrosophila. Nevertheless, a number of fascinating questions remain, including: (i) Why are these invertebrate proteins all of approx. 700–800 kDa? In terms of sarcomeric organization, what is the significance of this size? (ii) Why do three of these proteins consist of a mixture of Ig and Fn domains, whereas UNC-89 contains only Ig domains? This is even more interesting because the structures of Ig and Fn domains are very similar (118). What is the significance of the repeating pattern of groups of Ig and Fn domains (e.g. Fn-Fn-Ig)? (iii) How are twitchin and the synchronous muscle isoform of projectin situated on the surface of thick filaments? That is, do they form polymers or are they located at discrete locations with intervening gaps? (iv) What is the mechanism by which the fundamentally similar projectin isoforms get localized to different sarcomeric locations? (v) If the data onAplysia twitchin can be extended to the muscles of other invertebrates, what is the mechanism by which twitchin inhibits the rate of relaxation? How does phosphorylation of twitchin relieve this inhibition? (vi) What are the substrates for the protein kinase domains of nematode twitchin and insect projectin? If rMLCs are indeed the substrates, how would and why does this phosphorylation take place for the IFM isoform of projectin, which resides primarily in the I band? If rMLCs are the substrates, given the stoichiometry of approx. 1∶50 for twitchin:myosin, and the likely fixed position of twitchin along the thick filament, how does phosphorylation of just a few rMLCs result in a physiological effect (e.g. inhibition of relaxation)? What is the true activator for the twitchin and projectin kinases? (vii) How does UNC-89 participate in M-line assembly? (viii) What are the biochemical and physiological functions of intestinal brush border twitchin? A number of investigators will enjoy pursuing these and other questions for some time in the future.     

Five statistical questions about the tree of life

Stochastic modeling of phylogenies raises five questions that have received varying levels of attention from quantitatively inclined biologists. 1) How large do we expect (from the model) the ratio of maximum historical diversity to current diversity to be? 2) From a correct phylogeny of the extant species of a clade, what can we deduce about past speciation and extinction rates? 3) What proportion of extant species are in fact descendants of still-extant ancestral species, and how does this compare with predictions of models? 4) When one moves from trees on species to trees on sets of species (whether traditional higher order taxa or clades within PhyloCode), does one expect trees to become more unbalanced as a purely logical consequence of tree structure, without signifying any real biological phenomenon? 5) How do we expect that fluctuation rates for counts of higher order taxa should compare with fluctuation rates for number of species? We present a mathematician's view based on an oversimplified modeling framework in which all these questions can be studied coherently.     

Developments toward large-scale bacterial bioprocesses in the presence of bulk amounts of organic solvents

Many pseudomonads and other bacteria can grow on aliphatic and aromatic hydrocarbons that occur in the environment. We are examining the potential of such organisms as biocatalysts for the oxidation of a variety of substituted aliphatic and aromatic compounds. To attain a high production rate of oxidation products via such biotransformations, we have focused on two-liquid phase culture systems. In these systems, cells are grown in liquid media consisting of an aqueous phase containing water-soluble growth substrates and droplets of a water-immiscible organic solvent containing bioconversion substrates and products. For industrial applications of such two-liquid phase processes, several questions remain. What are the maximum rates at which apolar compounds can be transferred from the apolar phase to cells growing in the aqueous phase, i.e., what are the maximum space-time yields attainable in two-liquid phase fermentations under practical conditions? What does an efficient downstream processing of two-liquid phase medium involve? What safety regimes should be considered in working with flammable organic solvents? Can elevated pressure be used to increase oxygen transfer? Based on answers to these questions, we have recently developed a high-pressure, explosion-proof bioreactor system with Bioengineering AG (Wald, Switzerland), which will be installed in our pilot plant and used to explore two-liquid phase bioconversions at a pilot scale.     

Yeast programmed cell death: an intricate puzzle

Yeasts as eukaryotic microorganisms with simple, well known and tractable genetics, have long been powerful model systems for studying complex biological phenomena such as the cell cycle or vesicle fusion. Until recently, yeast has been assumed as a cellular 'clean room' to study the interactions and the mechanisms of action of mammalian apoptotic regulators. However, the finding of an endogenous programmed cell death (PCD) process in yeast with an apoptotic phenotype has turned yeast into an 'unclean' but even more powerful model for apoptosis research. Yeast cells appear to possess an endogenous apoptotic machinery including its own regulators and pathway(s). Such machinery may not exactly recapitulate that of mammalian systems but it represents a simple and valuable model which will assist in the future understanding of the complex connections between apoptotic and non-apoptotic mammalian PCD pathways. Following this line of thought and in order to validate and make the most of this promising cell death model, researchers must undoubtedly address the following issues: what are the crucial yeast PCD regulators? How do they play together? What are the cell death pathways shared by yeast and mammalian PCD? Solving these questions is currently the most pressing challenge for yeast cell death researchers.     

How to deal with neglected tropical diseases in the light of an African ethic

Many countries in Africa, and more generally those in the Global South with tropical areas, are plagued by illnesses that the wealthier parts of the world (mainly ‘the West’) neither suffer from nor put systematic effort into preventing, treating or curing. What does an ethic with a recognizably African pedigree entail for the ways various agents ought to respond to such neglected diseases? As many readers will know, a characteristically African ethic prescribes weighty duties to aid on the part of those in a position to do so, and it therefore entails that there should have been much more contribution from the Western, ‘developed’ world. However, what else does it prescribe, say, on the part of sub‐Saharan governments and the African Union, and are they in fact doing it? I particularly seek to answer these questions here, by using the 2013‐16 Ebola crisis in West Africa to illustrate what should have happened but what by and large did not.     

Curbing autophagy and histone deacetylases to kill cancer cells

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.     

Curbing autophagy and histone deacetylases to kill cancer cells

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.     

Valéry et l’automatisme psychologique

While the name of Paul Valery is usually associated with that of Leonardo da Vinci or the character, Monsieur Teste, most people are unaware of Valery’s posthumous work, which he considered the most significant of his writings: Cahiers/Notebooks, a voluminous and dense work, in its dimensions (around 30,000 pages) as well as in what it discloses about the daily ponderings of a man who considered himself — in spite of himself — a philosopher. It is a multifaceted work, touching on all fields of thought: history, politics, and the sciences, in all their diversity, from mathematics to physics, and biology to the theory of evolution. Neither philosophy, nor theology, nor linguistics is left in the dark. But he clearly expressed a preference for psychology and, more specifically, the question of mental function: What is it to think? How does the mind link ideas? What is abstraction? What role do feelings play? What is the relationship between the mind and the machine? Must we concede the existence of the unconscious mind. These are some of the questions we address in elucidating the concept of automatism through what makes it work as well as its failings.     

Signaling pathway of autophagy associated with innate immunity

Autophagy has recently been shown to be an important component of the innate immune response. The signaling pathways leading to activation of autophagy in innate immunity are not well studied. Our recent study shows that Toll-like receptor 4 (TLR 4) serves as an environmental sensor for autophagy. We define a new molecular pathway in which lipopolysaccharide (LPS) induces autophagy in human and murine macrophages by a pathway regulated through Toll-interleukin 1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-dependent, myeloid differentiation factor 88 (MyD88)-independent TLR4 signaling. Receptor-interacting protein (RIP1) and p38 mitogen-activated protein-kinase (MAPK) are downstream components of this pathway. This signaling pathway does not affect cell viability, indicating that it is distinct from an autophagic death signaling pathway. We further show that LPS-induced autophagy can enhance mycobacterial co-localization with the autophagosomes. The above study raises important questions. (1) What is the complete signaling pathway for LPS-induced autophagy? (2) Does TLR3 mediate autophagy? (3) What are the mechanisms that determine whether autophagy acts as a pro-death or pro-survival pathway? (4) What are the physiological functions of LPS-induced autophagosomes? Future studies examining the above questions should provide us with important clues as to how autophagy is regulated in innate immunity, and how autophagy can be utilized in pathogen clearance.     

Functional role of the Werner syndrome RecQ helicase in human fibroblasts

Werner syndrome is an autosomal recessive human genetic instability and cancer predisposition syndrome that also has features of premature aging. We focused on two questions related to Werner syndrome protein (WRN) function in human fibroblasts: Do WRN‐deficient fibroblasts have a consistent cellular phenotype? What role does WRN play in the recovery from replication arrest? We identified consistent cell proliferation and DNA damage sensitivity defects in both primary and SV40‐transformed fibroblasts from different Werner syndrome patients, and showed that these defects could be revealed by acute depletion of WRN protein. Mechanistic analysis of the role of WRN in recovery from replication arrest indicated that WRN acts to repair damage resulting from replication arrest, rather than to prevent the disruption or breakage of stalled replication forks. These results identify readily quantified cell phenotypes that result from WRN loss in human fibroblasts; delineate the impact of cell transformation on the expression of these phenotypes; and define a mechanistic role for WRN in the recovery from replication arrest.     

ON THE DIFFERENTIAL CYTOTOXICITY OF ACTINOMYCIN D

Actinomycin D (AMD) at concentrations that inhibit cellular RNA synthesis by 85% or more causes an acute phase of lethal cell degeneration in HeLa cultures beginning as early as 3 hr after drug exposure, resulting in the nearly complete loss of viable cells by 12 hr. The loss of cells during this acute phase of lethality is closely dose dependent. Vero, WI38, or L cells are not susceptible to this early acute cyto-intoxication by AMD, and may begin to die only after 1–2 days. Differential susceptibility to acute cyto-intoxication by AMD, or other inhibitors of RNA synthesis (daunomycin or nogalamycin), among different types of cultured cells is analogous to that observed in vivo in certain tissues and tumors, and cannot be accounted for by differences in the effect of AMD on RNA, DNA, or protein syntheses, or by the over-all loss of preformed RNA. Actinomycin D in a dose that inhibits RNA synthesis causes an equivalent loss of the prelabeled RNA in all the cell types studied. Inhibition of protein synthesis with streptovitacin A or of DNA synthesis with hydroxyurea does not cause acute lethal injury in HeLa cells as does inhibition of RNA synthesis. Furthermore, since Vero or L cells divide at about the same rate as HeLa cells, no correlation can be drawn between the rate of cell proliferation and susceptibility to the cytotoxicity of AMD. Susceptibile cells are most vulnerable to intoxication by AMD in the G₁-S interphase or early S phase. Inhibition of protein synthesis (which protects cells against damage by other agents affecting DNA) does not protect against AMD-induced injury. Although HeLa cells bind more AMD at a given dose than Vero or L cells, the latter cell types, given higher doses, can be made to bind proportionally more AMD without succumbing to acute cyto-intoxication. It is suggested that the differential susceptibility of these cell types to acute poisoning by AMD may reflect differences among various cells in the function or stability of certain RNA species not directly involved in translation whose presence is vital to cells. In HeLa cells, these critical species of RNA are presumed to have a short half-life.     

AIDS dementia and HIV-1-induced neurotoxicity: Possible pathogenic associations and mechanisms

AIDS Dementia Complex (ADC) is a syndrome of cognitive, behavioral, and motor deficits resulting from HIV-1 infection within the brain. ADC is characterized by variable degrees of neuronal cell death and gliosis that likely result, at least, in part from release of metabolic products, cytokines, and viral proteins from infected macrophages, although a unifying explanation for the neurological dysfunction has yet to be established. Major unanswered questions include: (i) do neurologic symptoms result from neuronal cell death and/or dysfunction in surviving neurons?; (ii) are viral genomic sequences determinants of neurotoxicity?; (iii) is HIV infection of neurons and astrocytes relevant to pathogenesis?, and (iv) what circulating factors within the brain affect neuronal cell survival and function? This review addresses the association between HIV-1 replication within the brain, production of potential neurotoxins and possible mechanisms of induction of neurotoxicity and neuronal dysfunction contributing to the pathogenesis of ADC.     

Interactions between intrinsic membrane protein and electric field. An approach to studying nerve excitability

We have approached the problem of nerve excitability through three questions: (a) What is the diagram for a channel? That is, what conformational states can the protein assume, and what transitions between these conformations are permitted? (b) What is the channel conductance associated with each conformation the channel can assume? (c) How do the rates for conformational transition depend upon membrane potential? These three questions arise from a standard statistical mechanical treatment of a nerve membrane containing several classes of identical, independent channels. Gating of channels, in this view, is associated with conformational changes of the channel protein, and it is assumed these conformations are distinct. The precise formulation of these questions is presented in terms of the theoretical treatment, and the approaches we have taken to answer the questions are indicated. Our present results indicate: Transition rates should depend exponentially on membrane potential over a limited voltage range, but probably will show a more complex dependence for extremes of the range; channels probably can take on only two conductances, open and shut, but more complicated situations are not entirely excluded; the diagram for a channel cannot be determined from standard voltage clamp data alone, but by studying gating currents and conductance fluctuations, it should be possible to select between alternative plausible physical mechanisms.     

Recognising and Translating Knowledge: Navigating the Political,Epistemological, Legal and Ontological

What does it mean to call something ‘knowledge’ today? What does this recognition or translation require? And what does it entrain? This introduction makes a novel synthesis of contributions to the Special Issue and advances observations regarding the ‘mythic’ qualities of intellectual property law, the precipitation of ‘nature’, and the importance of attending to what is lost when things and practices are also called ‘knowledge’. The papers cohere around a timely set of observations and critiques: critiques of the way the knowledge economy makes demands and defines expectations about value; of how intellectual property law lies behind and shapes exclusions, inclusions, and inequalities; of the ‘mythic’ status of assumptions informing laws about ownership; and the implicit hierarchy contained within types of knowledge as understood through the categories of western epistemology. By taking up effect rather than veracity and certainty, contributors leave the definition of knowledge to ethnographic subjects. That is, they attend to where and how things come to be called knowledge, and for what reasons, noticing how equivalences across practices, made for the purpose of creating the possibility of exchange value (and thus of encouraging circulation) does its work at the expense of multiple aspects, values, and relations that are also discernable in social processes that produce ‘knowledge’.     

Approche ph��nom��nologique du n��ant

In this article, we address the issue of nothingness on the basis of perceptive experience in its phenomenological dimension. Through an analysis of the visual perception and various types of memories related to it, we will try to answer three questions: what is phenomenological nothingness? What role does it play in the emergence of determinations within the consciousness? What are its relationships with disappearance and forgetting involved in all the stages of object donation in consciousness?     

Modulation of circuits underlying rhythmic behaviors

Summary What have we learned about behavior from neuromodulatory studies of the crustacean stomatogastric system? The emphasis of this paper has been on the analysis of one single class of behaviors (rhythmic) in terms of microcircuitry (synaptic connections between identified neurons). But in the general case, all behaviors result from the generation of spatio-temporal patterns by the central nervous system. How individual nerve cells interact with each other to produce such patterns is of fundamental interest. We know from work on simple networks that it is possible to link the circuitry of the nervous system with behavior in a precise way, and that instead of a large number of dedicated circuits, behaviors can be altered by chemically adjusting the functional properties of the neuronal elements. One circuit can be configured to perform a variety of different behaviors by activating neurons which contain neuromodulatory substances or in response to neurohormones circulating in the hemolymph. At present we know only a few of the ways neuromodulatory neurons are triggered to release their contents onto the neurons making up CPGs.The findings described here raise many questions. What are the parameters which control the distribution of neuromodulatory substances throughout the nervous system? What happens when more than one neuromodulator is present? At the cellular level, what mechanisms are involved in transforming each neuron from one functional state to another, and then how does the entire constellation of changes give rise to a new output? It is important to answer such questions in reduced networks, because there are presently no techniques available to answer them in the more complex networks of the brain. While there is no question that modulatory activity occurs in the brain, whether or not the principles which have been discovered by using simple invertebrate circuits scale up to vertebrate circuits remains an intriguing question.     

Is affordable and cost-effective assisted reproductive technology in low-income countries possible? What should we know to answer the question?

¹ Correspondence address. E-mail: j.d.f.habbema{at}erasmusmc.nl Decision making on infertility treatment in low-income countries(LIC) assumes answers to quite a few questions: how should theinfertility problem be defined? How often does infertility occur?What is the burden-of-disease of infertility? What is the incomein LIC, and what can be spend on health care? How cheap shouldIVF be in order to be accessible to a considerable part of thepopulation? With what alternative health interventions shouldinfertility treatment be compared? How cost-effective shouldIVF be in order to compete with those other interventions? Thesequestions will be discussed. The emphasis is on the situationin Sub-Saharan Africa (SSA). It is concluded that a place forART in a health care package is not straightforward. Many ofthe questions are not or only partially answered. Moreover,cheap and effective ART has yet to be developed and tested.From the limited evidence available for each of the questions,it could be calculated that an IVF cycle should cost between50 and 75 dollar in order to be a candidate for the inclusionin a health package in SSA. This estimate can easily changeconsiderably when in the future the calculations will be basedon thorough research. Thus, a targeted research programme foranswering the open questions, especially on quality-of-lifeimplications of infertility in different societies, is the preferredoption for facilitating the future evaluation of ART in LIC.