Cellular Analog Research Articles

(Very) Small Stem-like Cells in Human Cell Cultures

Very Small Embryonic-like Stem Cells (VSELSCs) and Very Small Cancer Stem Cells (VSCSCs) are fields of intensive research. Although the presence in vitro of VSELSC and VSCSC cellular stage analogs appear probable, it has yet to be published. Utilizing established human cell cultures with varying populations of primitive cells, stained with CD markers specific to primitive stages, in addition to a fluorescent DNA dye, and following histochemical processing, we have developed a cytological method for detecting Very Small Leukemic Stem-like Cells (VSLSLCs), Very Small Cancer Stem-like Cells (VSCSLCs), and VSELSCs. This detection provides an opportunity to advance research in these areas.

Behavioral characterization of Capn15 conditional knockout mice

Calpain 15 (CAPN15) is an intracellular cysteine protease belonging to the non-classical small optic lobe (SOL) family of calpains, which has an important role in development. Loss of Capn15 in mice leads to developmental eye anomalies and volumetric changes in the brain. Human individuals with biallelic variants in CAPN15 have developmental delay, neurodevelopmental disorders, as well as congenital malformations. In Aplysia, a reductionist model to study learning and memory, SOL calpain is important for non-associative long-term facilitation, the cellular analog of sensitization behavior. However, how CAPN15 is involved in adult behavior or learning and memory in vertebrates is unknown. Here, using Capn15 conditional knockout mice, we show that loss of the CAPN15 protein in excitatory forebrain neurons reduces self-grooming and marble burying, decreases performance in the accelerated roto-rod and reduces pre-tone freezing after strong fear conditioning. Thus, CAPN15 plays a role in regulating behavior in the adult mouse.

Molecular Docking and Analysis of In Silico Generated Ligands against SARS-CoV-2 Spike and Replicase Proteins

Purpose: The novel coronavirus also known as coronavirus disease 2019 (COVID-19), or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which broke out in the latter part of the year 2019, took the entire human race unawares. This sis due to its devastating health, social and economic consequences. In this study, the ability of some small molecules to interact with some SARS-CoV-2 proteins was investigated in silico for the purpose discovering molecules which can be employed in the areas COVID-19 diagnosis and treatment. Methodology: By way of molecular docking, a library of in silico generated ligands was docked to SARS-CoV-2 spike and replicase proteins to identify leads with propensity to bind them with high affinity. The identified leads proved to bind these proteins with stronger affinity than the native ligand aiding in their in vivo metabolic processes. Findings: It was observed that spike protein binds to its cellular receptor with binding affinity of -4.8Kcal/mol; it binds to a non cellular analogue with -5.4, while 4twy 3BL and 5n19 D03 bind spike protein with binding affinities of -7.3Kcal/mol each. They also bind replicase protein with -8.2 and -7.2 Kcal/mol respectively. 5c8s G3A and 2d2d ENB were identified as the most suitable leads for SARS-CoV-2 spike protein detection, while 3d62 959 and 1r4l XX5 were identified as leads with most suitable druglikeness against SARS-CoV-2. These findings indicate that the identified ligands can preferentially displace or inhibit binding of the viral proteins to their native endogenous ligands and that both cellular attachment through spike and ACE2 interaction, and viral replication process can both be inhibited by using just one of the substances identified. This study is part of efforts in finding non recombinant nucleic acid solutions to SARS-CoV-2 diagnosis and treatment. If these findings are implemented, they can enhance efficient detection of the virus antigens from biological samples. Conclusion: Identifying molecules that can interact with SARS-CoV-2 proteins could optimize diagnostic and therapeutic care for patients infected with the virus. Recommendation: Based on the study, 5c8s G3A and 2d2d ENB were identified as the most suitable leads that are favorably disposed for SARS-CoV-2 spike protein detection from biological samples. Also, 3d62 959 and 1r4l XX5 were identified as leads with most suitable drug likeness against SARS-CoV-2 based on the filters from SwissADME and Molinspiration cheminformatics and therefore deserve further in vitro and in vivo evaluations.

Inducible Tertiary Lymphoid Structures: Promise and Challenges for Translating a New Class of Immunotherapy.

Tertiary lymphoid structures (TLS) are ectopically formed aggregates of organized lymphocytes and antigen-presenting cells that occur in solid tissues as part of a chronic inflammation response. Sharing structural and functional characteristics with conventional secondary lymphoid organs (SLO) including discrete T cell zones, B cell zones, marginal zones with antigen presenting cells, reticular stromal networks, and high endothelial venues (HEV), TLS are prominent centers of antigen presentation and adaptive immune activation within the periphery. TLS share many signaling axes and leukocyte recruitment schemes with SLO regarding their formation and function. In cancer, their presence confers positive prognostic value across a wide spectrum of indications, spurring interest in their artificial induction as either a new form of immunotherapy, or as a means to augment other cell or immunotherapies. Here, we review approaches for inducible (iTLS) that utilize chemokines, inflammatory factors, or cellular analogues vital to TLS formation and that often mirror conventional SLO organogenesis. This review also addresses biomaterials that have been or might be suitable for iTLS, and discusses remaining challenges facing iTLS manufacturing approaches for clinical translation.

2D printed multicellular devices performing digital and analogue computation

Much effort has been expended on building cellular computational devices for different applications. Despite the significant advances, there are still several addressable restraints to achieve the necessary technological transference. These improvements will ease the development of end-user applications working out of the lab. In this study, we propose a methodology for the construction of printable cellular devices, digital or analogue, for different purposes. These printable devices are designed to work in a 2D surface, in which the circuit information is encoded in the concentration of a biological signal, the so-called carrying signal. This signal diffuses through the 2D surface and thereby interacts with different device components. These components are distributed in a specific spatial arrangement and perform the computation by modulating the level of the carrying signal in response to external inputs, determining the final output. For experimental validation, 2D cellular circuits are printed on a paper surface by using a set of cellular inks. As a proof-of-principle, we have printed and analysed both digital and analogue circuits using the same set of cellular inks but with different spatial topologies. The proposed methodology can open the door to a feasible and reliable industrial production of cellular circuits for multiple applications.

Development of Animal Models, Presenting Appropriate Analogues to Respective Human Systems

The main idea of the current study was directed to developed appropriate experimental animal models, imitating respective systems with the human origin, and giving a possibility when the last is not available, experiments about necessary applications to humans to be performed. So, an additional copy of oncogene Dcn1 in normal mouse embryonic stem cells (mESCs), was inserted, by appropriate recombinant DNA-constructs, based on the AAV DNA-genome. All derived genetically-manipulated cellular types were co-cultivated with early myeloid and lymphoid progenitors, derived from non-transfected mESCs in the presence of GM-CSF (for induction of initial stages of both myeloid and lymphoid differentiation), and subsequently, malignant antigens were added (about further phagocyte and plasmatic cell differentiation, respectively). The derived and selected mESCs, containing an additionally-inserted copy of oncogene Dcn1, which indicated preserved normal/non-malignant characteristics both in vitro and in vivo, presented appropriate experimental normal mouse cellular analog of the cited in the scientific literature human embryonic trophoblasts, immortalized by infection with virus SV40. Additionally, the results obtained showed a possibility about the expression of membrane receptor glycoproteins by non-lymphoid and non-myeloid cellular types inappropriate conditions. Also, the presented study demonstrated the importance of the blood-testes barrier (BTB) for the prevention of malignancy development in the experimental hamster Graffi tumor model. The role of bio-molecules, as well as of intra- and extra-cellular inter-molecular interactions in cascade regulatory mechanisms, inactivation of the differentiation of embryonic and adult stem/progenitor cells in normal types, as well as for suppression of malignant transformation, was suggested. The established analogy of the developed and investigated in the current study experimental animal models gives a possibility for their application about performing of specific experiments when the respective systems with human origin are not available.

Assisted Same-Sex Reproduction: The Promise of Haploid Stem Cells?

Same-sex couples, not unlike their heterosexual counterparts, would prefer having a genetically related child. However, assisted same-sex human reproduction has heretofore been deemed infeasible absent haploid cellular analogs of human gametes. Recent developments, however, may have overcome this limitation through the derivation of haploid embryonic stem cells (hapESCs). Undifferentiated, pluripotent, self-renewing, and stably haploid, hESCs have also displayed germline competence. It is in this capacity that murine hESCs, doubling up as de facto gametes, gave rise to bimaternal and bipaternal progeny. Herein we argue that assisted same-sex human reproduction, although potentially attainable at this time, is still years away from the clinic. In support of this perspective, we note the significant technical, regulatory, statutory, and societal hurdles that stand in the way of near-term implementation.

Effects of Noopept and Piracetam on Depression of the Acetylcholine-Induced Current in Common Snail Command Neurons

The actions of the nootropes noopept and piracetam on depression of the acetylcholine-induced current in a cellular analog of habituation were studied. Mathematical modeling of experimental curves and their analysis using previously obtained results on the effects of inhibitors of different protein kinases and protein phosphatases on acquisition of depression of the acetylcholine-induced current in a cellular analog of habituation clarified the intracellular processes and targets on which these agents act.

Measurement of Internal Implantation Strains in Analogue Bone Using DVC.

The survivorship of cementless orthopaedic implants may be related to their initial stability; insufficient press-fit can lead to excessive micromotion between the implant and bone, joint pain, and surgical revision. However, too much interference between implant and bone can produce excessive strains and damage the bone, which also compromises stability. An understanding of the nature and mechanisms of strain generation during implantation would therefore be valuable. Previous measurements of implantation strain have been limited to local discrete or surface measurements. In this work, we devise a Digital Volume Correlation (DVC) methodology to measure the implantation strain throughout the volume. A simplified implant model was implanted into analogue bone media using a customised loading rig, and a micro-CT protocol optimised to minimise artefacts due to the presence of the implant. The measured strains were interpreted by FE modelling of the displacement-controlled implantation, using a bilinear elastoplastic constitutive model for the analogue bone. The coefficient of friction between the implant and bone was determined using the experimental measurements of the reaction force. Large strains at the interface between the analogue bone and implant produced localised deterioration of the correlation coefficient, compromising the ability to measure strains in this region. Following correlation coefficient thresholding (removing strains with a coefficient less than 0.9), the observed strain patterns were similar between the DVC and FE. However, the magnitude of FE strains was approximately double those measured experimentally. This difference suggests the need for improvements in the interface failure model, for example, to account for localised buckling of the cellular analogue bone structure. A further recommendation from this work is that future DVC experiments involving similar geometries and structures should employ a subvolume size of 0.97 mm as a starting point.

Reproduction reimagined

Reproduction reimagined

Integration of energy homeostasis and stress by parvocellular neurons in rat hypothalamic paraventricular nucleus.

Central regulation of energy homeostasis and stress are believed to be reciprocally regulated, i.e. excessive food intake suppresses, while prolonged hunger exacerbates, stress responses in vivo. This relationship may be mediated by neuroendocrine parvocellular corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus that receive both stress- and feeding-related input. We find that hunger strongly and selectively potentiates, while re-feeding suppresses, a cellular analogue of a stress response induced by acute glucopenia in CRH neurons in rat hypothalamic slices. Neuronal activation in response to glucopenia was mediated synaptically, via the relative enhancement of glutamate over GABA input. These results illustrate how acute stress responses may be initiated in vivo and show that it is reciprocally integrated with energy balance via local hypothalamic mechanisms acting at the level of CRH neurons and their afferent terminals. Increased food intake is a common response to help cope with stress, implying the existence of a previously postulated but imperfectly understood, inverse relationship between the regulation of feeding and stress. We have identified components of the neural circuitry that can integrate these homeostatic responses. Prior fasting (∼24h) potentiates, and re-feeding suppresses, excitatory responses to acute glucopenia in about half of the corticotropin releasing hormone (CRH)-expressing, putatively neurosecretory, stress-related neurons in the paraventricular nucleus of the hypothalamus studied. Glucoprivation stress ex vivo resulted from a preferential relative increase in excitatory (glutamatergic) over inhibitory (GABAergic) inputs. Putative preautonomic cells were less sensitive to fasting, and showed a predominant inhibition to acute glucopenia. We conclude that hunger may sensitize hypothalamic stress responses by acting via local mechanisms, at the level of CRH neurons and their presynaptic inputs. Those mechanisms involve neither presynaptic ATP-sensitive potassium channels nor postsynaptic ATP levels.

Structural basis for substrate selectivity and nucleophilic substitution mechanisms in human adenine phosphoribosyltransferase catalyzed reaction

The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (SN2) with a mix of SN1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted SN2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates.

Change of phenotypic properties of escape mutants and readaptants of influenza virus A (H1N1)pdm09 under the influence of selected mutations in the molecule of hemagglutinin.

After the emergence and spread of pandemic H1N1 viruses in 2009, antigenic epitopes recognized by neutralizing antibodies against the hemagglutinin of influenza A/Moscow/01/09(H1N1)pdm09 viruses were studied. The purpose of the study was to obtain readapted variants of the virus from a low-virulent escapemutant that has an increased affinity of the avian and the human types cellular receptors compared to the wild type and the comparative study of their antigenic and receptor specificity. Viruses were accumulated in 10-day-old chicken embryos. The MAB panel against HA of influenza virus strain A/IIV-Moscow/01/09(H1N1)sw1 was used in the form of ascites fluids from mice. Immunization of mice, HI testing, elution of viruses from chicken erythrocytes, PCR and sequencing of readapted variants were performed by standard methods. The amino acid substitution A198E acquired in the process of readaptation leads to changes in the antigenic specificity. A correlation was found between a decrease in virulence of a low-virulent escape mutant associated with the substitution D190N in the hemagglutinin molecule and an increase in the hemagglutinating titer to inhibitors in normal mouse serum. Viruses with low affinity of cellular receptor analogs and carrying amino acid substitutions have an increased ability to elute from chicken erythrocytes. The results discuss the effect of mutations in the HA molecule of the influenza A(H1N1) pdm09 virus to the change in antigen specificity; virulence for mice, adsorption-elution at cellular receptors. A comparative study of the antigenic specificity and receptor-binding activity of the escape mutants was conducted for the hemagglutinin of the influenza virus A/Moscow/01/2009 (H1N1)swl, and the readapted variants obtained for one of the escape mutants with reduced virulence for mouse. Monitoring the pleiotropic effect of mutations in the hemagglutinin H1 molecule is necessary to predict variants of the virus with pandemic potential.

An Ultrafast Cellular Method for Matrix Multiplication

The author considers the ultrafast cellular method of matrix multiplication that deals with cellular submatrices, interacts with well-known matrix multiplication cellular methods, and minimizes by 12.5% the computing complexity of cellular analogs of well-known matrix multiplication algorithms generated on their basis. The interaction of the ultrafast cellular method with the unified cellular method of matrix multiplication provides the highest (as compared with well-known methods) percentage (equal to 45.2%) of minimization of multiplicative, additive, and overall complexities of well-known matrix multiplication algorithms. The computing complexity of the ultrafast method is estimated using the models of getting cellular analogs of the traditional matrix multiplication algorithm.

Biodynamics: A novel quasi-first principles theory on the fundamental mechanisms of cellular function/dysfunction and the pharmacological modulation thereof.

Cellular function depends on heterogeneous dynamic intra-, inter-, and supramolecular structure-function relationships. However, the specific mechanisms by which cellular function is transduced from molecular systems, and by which cellular dysfunction arises from molecular dysfunction are poorly understood. We proposed previously that cellular function manifests as a molecular form of analog computing, in which specific time-dependent state transition fluxes within sets of molecular species (“molecular differential equations” (MDEs)) are sped and slowed in response to specific perturbations (inputs). In this work, we offer a theoretical treatment of the molecular mechanisms underlying cellular analog computing (which we refer to as “biodynamics”), focusing primarily on non-equilibrium (dynamic) intermolecular state transitions that serve as the principal means by which MDE systems are solved (the molecular equivalent of mathematical “integration”). Under these conditions, bound state occupancy is governed by kon and koff, together with the rates of binding partner buildup and decay. Achieving constant fractional occupancy over time depends on: 1) equivalence between kon and the rate of binding site buildup); 2) equivalence between koff and the rate of binding site decay; and 3) free ligand concentration relative to koff/kon (n · Kd, where n is the fold increase in binding partner concentration needed to achieve a given fractional occupancy). Failure to satisfy these conditions results in fractional occupancy well below that corresponding to n · Kd. The implications of biodynamics for cellular function/dysfunction and drug discovery are discussed.

Catalyst: On Organic Systems Chemistry and Origins Research

Yitzhak Tor is a professor of chemistry and biochemistry and the George W. and Carol A. Lattimer Professor at the University of California, San Diego. He earned his doctorate degree at the Weizmann Institute of Science (1990) and was a postdoctoral fellow at the California Institute of Technology (1990–1993). His research focuses on the chemistry and biology of nucleosides, nucleotides, and nucleic acids as well as the development of cellular delivery agents and fluorescent nucleoside analogs. He is the editor-in-chief of Perspectives in Medicinal Chemistry and Organic Chemistry Insights .

Cell-Specific PKM Isoforms Contribute to the Maintenance of Different Forms of Persistent Long-Term Synaptic Plasticity.

Multiple kinase activations contribute to long-term synaptic plasticity, a cellular mechanism mediating long-term memory. The sensorimotor synapse of Aplysia expresses different forms of long-term facilitation (LTF)-nonassociative and associative LTF-that require the timely activation of kinases, including protein kinase C (PKC). It is not known which PKC isoforms in the sensory neuron or motor neuron L7 are required to sustain each form of LTF. We show that different PKMs, the constitutively active isoforms of PKCs generated by calpain cleavage, in the sensory neuron and L7 are required to maintain each form of LTF. Different PKMs or calpain isoforms were blocked by overexpressing specific dominant-negative constructs in either presynaptic or postsynaptic neurons. Blocking either PKM Apl I in L7, or PKM Apl II or PKM Apl III in the sensory neuron 2 d after 5-hydroxytryptamine (5-HT) treatment reversed persistent nonassociative LTF. In contrast, blocking either PKM Apl II or PKM Apl III in L7, or PKM Apl II in the sensory neuron 2 d after paired stimuli reversed persistent associative LTF. Blocking either classical calpain or atypical small optic lobe (SOL) calpain 2 d after 5-HT treatment or paired stimuli did not disrupt the maintenance of persistent LTF. Soon after 5-HT treatment or paired stimuli, however, blocking classical calpain inhibited the expression of persistent associative LTF, while blocking SOL calpain inhibited the expression of persistent nonassociative LTF. Our data suggest that different stimuli activate different calpains that generate specific sets of PKMs in each neuron whose constitutive activities sustain long-term synaptic plasticity.SIGNIFICANCE STATEMENT Persistent synaptic plasticity contributes to the maintenance of long-term memory. Although various kinases such as protein kinase C (PKC) contribute to the expression of long-term plasticity, little is known about how constitutive activation of specific kinase isoforms sustains long-term plasticity. This study provides evidence that the cell-specific activities of different PKM isoforms generated from PKCs by calpain-mediated cleavage maintain two forms of persistent synaptic plasticity, which are the cellular analogs of two forms of long-term memory. Moreover, we found that the activation of specific calpains depends on the features of the stimuli evoking the different forms of synaptic plasticity. Given the recent controversy over the role of PKMζ maintaining memory, these findings are significant in identifying roles of multiple PKMs in the retention of memory.

Synchronization of In Vitro Maturation in Porcine Oocytes.

When removed from the follicles, during the 44 h process of in vitro maturation (IVM) fully grown porcine oocytes resume meiosis spontaneously from the late diplotene stage of the first meiotic prophase and proceed to the metaphase-II (MII) stage at which they remain arrested until fertilization. However, the spontaneous resumption may start at various times causing heterogeneity in the nuclear stage and also in cytoplasmic characteristics within a population. Those oocytes that reach the MII stage earlier than others undergo an aging process which is detrimental for further embryo development. The synchronization of nuclear progression of porcine oocytes can be achieved by a transient inhibition of meiotic resumption during the first 20-22 h of IVM by the elevation of intracellular levels of cyclic adenosine monophosphate (cAMP) using the cellular membrane-permeable analog of cAMP, dibutyryl cyclic AMP. A simple and efficient protocol for such treatment is described below.

Persistent Associative Plasticity at an Identified Synapse Underlying Classical Conditioning Becomes Labile with Short-Term Homosynaptic Activation.

Activity-dependent changes in neural circuits mediate long-term memories. Some forms of long-term memories become labile and can be reversed with specific types of reactivations, but the mechanism is complex. At the cellular level, reactivations that induce a reversal of memory must evoke changes in neural circuits underlying the memory. What types of reactivations induce a labile state at neural connections that lead to reversal of different types of memory? We find that a critical neural connection in Aplysia, which is modified with different stimuli that mediate different types of memory, becomes labile with different types of reactivations. These results provide insights for developing strategies in alleviating maladaptive memories accompanying anxiety disorders.

Multidimensional traveling waves in the Allen–Cahn cellular automaton

Ultradiscretization is a limiting procedure transforming a given difference equation into a cellular automaton. The cellular automaton constructed by this procedure preserves the essential properties of the original equation, such as the structure of exact solutions for integrable equations. In this article, a cellular automaton analog of the multidimensional Allen–Cahn equation which is not an integrable system is constructed by the ultradiscretization. Moreover, the traveling wave solutions for the resulting cellular automaton are given. The shape, behavior and stability of the solutions in ultradiscrete systems are similar to those in continuous systems.