Cell's with an Index like Google?

Its been a while since I last wrote about DNA repeats or their RNA descendants. In that time advanced research has emerged relating repeats to increasing numbers of viral or other disease. Generally the repeats of interest here can be either long or short sequences of nucleotides that from part of an unspliced gene. Logically, counts of long sequences that repeat would be less than short sequences, but when normalized to their respective nucleotide lengths the indexed results can shift the relative order of repeating sequences quite dramatically.

In most knowledge systems repeats in low level data present redundancy and opportunity to improve efficacy in local or global upstream processes acting on that data. We see this in the structure of efficient alphabets that had a significant impact on whether or not a language survived continuous use. Why use ten words when precise meaning, including abstracts can be derived from three. Or why alpha when, at least for some period in the language history alphanumeric made it more effective? 

Search engines reduce their primary index to the least redundant data set used to drive efficient data access by upstream requests and processes to satisfy any query. However, at the storage level, data redundancy is permitted because energy efficiency is gained. Similarly genetic DNA is massively redundant. Redundant data stores can make highly indexed systems more efficient because frequently accessed data elements are more accessible at multiple locations and parallel processes can more efficiently satisfy upstream requests.

Repetitive sequences constitute 50%–70% of the human genome. Some of these can transpose positions, these transposable elements (TE's) are DNA transposons and retrotransposons. The latter are predominant in most mammals and can be further divided into long terminal repeat (LTR)-containing endogenous retrovirus transposons and non-LTR transposons including short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs). The most abundant subclass of SINEs comprises primate-specific Alu elements in human with more abundant GC-rich DNA. Humans have up to 1.4 million copies of these repeats, which constitute about 10.6% of the genomic DNA. Long interspersed element-1 (LINE1 or L1), are abundant in AT-rich DNA, constitute 19% of the human genome and make up the largest proportion of transposable element-derived sequences.

Most TE classes are primarily involved in reduced gene expression, but Alu elements are associated with up regulated gene expression. Intronic Alu elements are capable of generating alternative splice variants in protein-coding genes that illustrate how Alu elements can alter protein function or gene expression levels. Non-coding regions were found to have a great density of TEs within regulatory sequences, most notably in repressors. TEs have a global impact on gene regulation that indicates a significant association between repetitive elements and gene regulation.

In liquid systems, phase separation is one of the most fundamental phase transition phenomena and ubiquitous in nature. De-mixing of oil and water in salad dressing is a typical example. The discovery of biological phase separation in living cells led to the identification that phase-separation dynamics are controlled by mechanical relaxation of the network-forming dense phase, where the limiting process is permeation flow of the solvent for colloidal suspensions and heat transport for pure fluids. The application of this derived governing universal law is a step to understanding and defining the liquid biological indexing equivalence of data-processing systems and inherent genetic redundancy.

Repeats have been widely implicated. In plant immunity a TE has been domesticated through histone marks and generation of alternative mRNA isoforms that were both directly linked to immune response to a particular pathogen. p53 transcription sites evolved through epigenetic methylation, deamination and histone regulation that constituted a universal mechanism found to generate various transcription-factor binding sites in short TE's or Alu repeats. In disease cytoplasmic synthesis of Alu cDNA was implicated in age related macular degeneration and there is transient increase of nearly 20-fold in the levels of Alu RNA during stress, viral infection and cancer.

In chromosomal DNA, each sequence, relative to its length may conveniently describe a phase-separated indexed location and method for discovery. Repeats within genetic DNA may present precisely sensitive phase-separated guidance to drive histone, epigenetic and transcription factors to specific genetic locations at the cells' 'end-of-line' from where the genetic response to upstream membrane bound changes begin.

 

Systolic Blood Pressure and Innate Immunity vs. the Cancer Brain

Participants with a valid heart disease phenotype (atherosclerosis) were identified in a MESA blood pressure analysis conducted over 10 years. The valid group varied from 770 to 1113 patients from whom further blood analysis queried a primary and exploratory hypothesis of immune cell subsets. Four statistically significant innate cell subsets were discovered to be associated with Systolic blood pressure (SBP); Natural Killer (NK) cells, gamma delta T cells and classical monocytes.

Separately, an analysis of 7017 individuals from 6 international studies of gene expression signatures for SBP, diastolic blood pressure (DBP) and hypertension (HTN) found 7717 genes of which 34 were most differentialy expressed. Enrichment analysis for the systolic and diastolic gene group's associated to NK cell mediated cytotoxicity and 13 other pathways including antigen processing and inflammatory response, pointing strongly to innate and adaptive immunity. MYADM was the only gene identified for all groups SBP, DBP and HTN.

MYADM controls endothelial barrier function through ezrin, radixin, and moesin (ERM)-dependent regulation of ICAM-1 expression. ERM expression is required for ICAM-1 expression in response to MYADM suppression or TNF-α. ICAM-1 is a paradigmatic adhesion receptor that regulates leukocyte adhesion together with integrin LFA-1. This connection between endothelial membrane and cortical actin cytoskeleton appears to modulate the inflammatory response at the blood tissue barrier. 

Pressure overload activates the sympathetic nervous system (SNS) and up-regulates p53 expression in the cardiac endothelium and in bone marrow (BM) cells. Increased p53 expression promotes endothelial-leukocyte cell adhesion and initiates inflammation in cardiac tissue, which exacerbates systolic dysfunction. SNS activates, at least by significant increase of circulating norepinephrine (NE), which up-regulates p53 expressions, while forced expression of p53 increased ICAM-1 expression. 

On endothelial cells SNS is mediated via catecholamine-β2-adrenergic signaling, which up-regulates the production of reactive oxygen species (ROS), activates p53 and induces cellular senescence. Immune cells, including macrophages, monocytes, NK cells, B and T cells express the β2-adrenergic receptor and catecholamine. During pressure overload, NE cultured macrophages up-regulated p53 expression, whereas introduction of p53 increased Itgal (LFA-1) expression (which binds ICAM-1). Treatment with NE increased ROS, which was attenuated after inhibition of β2- adrenergic signaling in macrophages. Endothelial cell–macrophage interaction via NE-ROS-p53 signaling induces up-regulation of adhesion molecules, thus contributing to cardiac inflammation and systolic dysfunction.

During hypertension the vascular endothelium activates monocytes, in part through ROS by a loss of nitric oxide (NO) signaling, increased release of IL-6, hydrogen peroxide and a parallel increase in STAT activation in adjacent monocytes. NO inhibits formation of intermediate monocytes and STAT3 activation. Humans with hypertension have increased intermediate and non-classical monocytes and  intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells, and macrophages with activated STAT3.

A senescence-associated secretory phenotype (SASP) was induced in epithelial cells after DNA damage of sufficient magnitude. In premalignant epithelial cells SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy by a paracrine mechanism that largely depended interleukin (IL)-6 and IL-8. Strikingly, loss of p53 and gain of oncogenic RAS exacerbated the pro-malignant activities. This suggests a cell-non-autonomous mechanism by which p53 can restrain and oncogenic RAS can promote the development of age-related cancer by altering the tissue microenvironment. Oncogenic signaling pathways inhibit the p53 gene transcription rate through a mechanism involving Stat3, which binds to the p53 promoter in vitro and in vivo. Blocking Stat3 in cancer cells up-regulates expression of p53, leading to p53-mediated tumor cell apoptosis. 

Induced stretch or stretch from pressure overload may engage a non-autonomous, p53 centric micro-mechanical mechanism that escalates or deescalates innate responses against cells functioning outside the mechanical ranges that macrophages or NK cells permit. Thus, the neuro-immune extension through SNS signaling, may begin with circulating blood pressure or stretch promoted through inflammation. 

Genetic Eruption and p53 Response!

L1 are a class of transposable DNA elements found in 17% of the genome that are evolutionarily associated with primitive viral origins. Around 100 have retained the ability to retrotranspose. Without restraint they can interrupt the genome through insertions, deletions, rearrangements, and copy number variations. L1 activity has contributed to instability and evolution of genomes, and is tightly regulated by DNA methylation, histone modifications, and piRNA. They can further impact genome variation by mispairing and unequal crossing-over during meiosis due to its repetitive DNA sequences. Indeed, meiotic double-strand breaks are the proximal trigger for retrotransposon eruptions as highlighted in animals lacking p53.

189 gastrointestinal cancer patients across three cancer types: 95 stomach, colorectal esophageal were examined for any aberration in DNA repair pathways that could be associated with L1 retro-transposition. Out of 15 DNA repair pathways, only the TP53 repair pathway showed a significant association. L1 retro-transposition is inversely correlated with expression of immunologic response genes including interferons. Frequent TP53 mutations in tumors with a higher load of L1 insertions suggest the critical role of TP53 in restricting retrotransposons as a guardian of L1 expression and cancer immunity.

A screen of 172 open reading frames (orfs), of unknown genetic function across several human viruses was designed to discover novel interactions with p53. The orfs encoded viral proteins, miRNA's and lncRNA's. The ORFEOME project was based on the hypothesis that every virus should encode some functions that interfere with the p53 signaling network. The methods present a broad net by screening for interactions without necessarily defining how interactions arise.

The DNA damage response (DDR) pathway stabilizes p53 leading to increased nuclear relocation, binding to p53 response elements, rearrangement of chromatin and transcription of p53 target genes. Any of the multiple p53 related interactions along the way is a potential target of translated viral proteins on the function of p53. 

p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling by the induction of genes containing IFN-stimulated response elements. p53 also contributed to an increase in IFN release from infected cells. This p53-dependent enhancement of IFN signaling is dependent to a great extent on p53 activation and transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Thus p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

p53 likely cooperates with histone and DNA methylation to silence specific retroelements. In the zebrafish model, it was shown that p53-dependent H3K9me3 methylation, in the promoter region of a synthetic human LINE1 element mapped to a known p53-binding site. Some evidence in human cell lines suggests that p53 can physically interact with both H3K9 tri-methyltransferases and DNA methyltransferases. In basal stress-free conditions, unacetylated p53 is pre-bound to many target genes together with SET - a repressor protein, which mediates repression of p53 target genes. Additionally, p53 as a master regulator of transcription might regulate gene expression of key epigenetic or piRNA factors. 

Through L1's we get a sense of p53's interconnectedness to DNA damage, viral replication, cancer and immunity. In a way we can sympathize with it, especially when overloaded by viral infiltration and eruption. Its understandable how, under those conditions double stranded DNA breaks and pathway impediments compromise its ability to be guardian of the genome!

Natural Killers at the Neuro-Immune Axis!

Much has been said about the role Natural Killer (NK) cells play in positively and negatively influencing events in tissues and cells. Summarized facts about the healthy state of NK cells in humans and animals explain how innate immune cells, including NK cells differ from adaptive immune cells. One significant feature of NK cells is that they can act independently of MHC antigen presentations and that makes them tantalizing, but enormously challenging for scientists seeking to embrace their  influence over cells and their killing capabilities.

The variety and combination of inhibitory and activating receptors differentially expressed by as many as 30,000 human NK cell subsets makes heterogeneity difficult to relate across different conditions, organs and tissue types. Notwithstanding, positive rates of overall patient survival resoundingly corelated to the presence of as few as one NK cell infiltrating a tumor in a microscopic field.  

Innate immune cells including NK and macrophages have also been directly tied to conditions of neurological pain and more specifically to afferent and efferent fibers that signal through the vagus nerve. In these models at the immune-neurological interface similarities exists and both organs must interact for proper function. 

In each of these organs communication is mediated by direct cellular contact eg. synapse formation and via soluble mediators like cytokines or neurotransmitters that also communicate bi-directionally between cells of each system. The nervous and immune systems can influence each other’s activity because immune cells express neurotransmitter receptors, and neurons express cytokine receptors. Immune cells can synthesize and release neurotransmitters themselves, thus using neurotransmitter-mediated pathways via autocrine and paracrine mechanisms. This may indicate that NK cells extend nerve end signaling further into tissues and at a cellular level. 

A recent paradigm in physiology describes the existence of neuro-immune cell units, at an organ-tissue level and identifies the enormous complexity inherent in this globally unifying approach that also connects neuro-immune-gut, at least in Parkinson's disease.

Parkinson’s is a brain disorder where certain nerve cells slowly die and symptoms worsen. The risk of developing the condition increases with age, but in certain patients the illness is caused by defects in two proteins, PINK1 and parkin. NK cells are capable of homing to the central nervous system in neurological disorders that exhibit exacerbated inflammation and inhibit hyperactivated microglia. Recently, a study demonstrated that NK cells scavenge alpha-synuclein aggregates and systemic depletion of NK cells results in exacerbated neuropathology in a mouse model of alpha-synucleinopathy, making NK cells highly relevant in Parkinson’s disease.

We recently described a mechanism by which the sentinel state of NK cells is impaired and suggested the senescent phenotype, induced by age related mitophagy could be the primary cause. Increase in mitophagy (mitochondrial autophagy) is age-dependent and abrogated by PINK1 or parkin deficiency suggesting, in Parkinson's disease compromised mitophagy is associated with neurological degeneration. Further  PINK1 and Parkin, which are regulated by p53 specifically repress mitochondrial antigen presentation of both MHC classes. Therefore, excessive PINK1 or parkin increases rates of NK cell mitophagy and repress the presentation of mitochondrial antigens for MHC classes at the axis of this neuro-immune related disease.

The healthy state of NK cells at the axis of neuro-immune systems may indeed have more far reaching implications for the future of human diseases and therapies.

 

Natural Killers Linked to Overall Survival in Cancer

A meta analysis of tumor samples, collected between 1973 and 2016, in 53 studies confirmed overall survival (OS) correlated with Natural Killer cell infiltration into solid tumors. The number of NK cells infiltrating solid tumors, including those considered “highly ”infiltrated was relatively low, compared with other immune populations. Notwithstanding, the presence of a single NK cell, within a high powered microscopic field was associated with significantly improved OS and disease free survival in colorectal cancer, HER2 + breast cancer and hepatocellular carcinoma.

The finding supports the prospect that single tumor infiltrating NK cells, in a sampled tissue can be determinative for OS. By inference a single tumor infiltrating NK cell or cells possess characteristics that are relative to OS and beneficial to patient.  

NK cell surface receptors are densely varied defining at least 30,000 unique NK cell populations within each individual. NK cell classifications, relative to tumor infiltration and OS is enormously complex, especially at this scale and present definitions of activating and inhibiting receptor combinations underwhelm. To identify NK cells that have infiltrated or may be capable of infiltrating a patient tumor to improve OS we focused on biopsied tumor tissue selections whether or not they include NK cells.

Our work is with two tumor types in humanized mice. Multiple sections of each tumor were resected and divided into multiple parts for coculture with allogenic naïve, IL2 and probiotic enhanced NK cells and for DNA sequencing. After coculture NK cell cytotoxicity and other detailed measures resulting from each resected section and from single cells were assessed. Presently sequencing of DNA from each resected, divided section (pre-coculture) is focused on comparisons derived from TP53.

In the final stage NK cells will be cocultured with resected tumor tissue and will be made to challenge new tumor tissue and single cells, from the resected tumor from which the NK coculture was derived. The objective will be whether Codondex analysis of TP53 DNA sequencing can predict the most successful tumor tissue candidates based upon the most effective cocultured NK cell challenge to the tumor derived tissue or cells. 

If Codondex algorithm is found to identify a direct or indirect logic for tissue or cell selection that is effective in vitro our work will continue to next stage in vivo testing and analysis on similar grounds. 

  

 

Not Only A Killer A System for Killing!

The next time you're out exercising, spare a thought for your busy mitochondria. NASA scientists just reported mitochondria as the key to health problems in space.

Natural killer (NK) cells can extend membrane probes into cells or pathogens. These are loaded with granulysin (GNLY) to penetrate and perforin (PFN) to kill intracellular bacteria or protozoa and can lyse entire cells. The probes can also transfer healthy mitochondria to apoptotic cardiomyocytes (and other cells) in need of mitochondrial transfer. Uterine NK cells of the decidua send probes into trophoblasts to selectively kill intracellular Listeria monocytogenes without killing the trophoblast host. Stressed cells, moving toward apoptosis can behave similarly, but in reverse shooting out nanoprobes to proximal cells seeking cooperation and urgent mitochondrial transfers including to cancer cells.

A meta-analysis of gene expression signatures for blood pressure and hypertension in 7017 individuals from 6 international studies found of 7717 genes, 34 were most differentialy expressed including GNLY. Enrichment analysis for the diastolic and systolic gene group's associated strongly with NK cell mediated cytotoxicity and 13 other pathways including antigen processing and inflammatory response.

Formation of membrane probes or tubes, in which mitochondria travel and establishment of intracellular mitochondrial networks in the peripheral zone of cells require Kinesin-1 heavy chain (KIF5B). KIF5B is also required for female meiosis (oogenesis) and proper chromosomal segregation in mitotic cells and modulates central spindle organization in late-stage cytokinesis in chondrocytes.

A study of centromere heterochromatin (connected with central spindle) surprisingly showed that distant euchromatic regions, enriched in repressed methylated genes also interacted with the hierarchical organization of centromeric DNA. These 3D spatial interactions (at a distance) are likely mediated by liquid-like fusion events and can influence the health of individuals. Repressed gene's were identified as transposable elements, sequences often associated with pathogenic DNA insertions that have been persistently retained.  

KIF5B is an interaction partner of ADP-ribosylation factor-like 8b (Arl8b), which is required for NK cell–mediated cytotoxicity that drives polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between NK and target cells. Silencing experiments that led to failure of MTOC-lytic granule polarization suggest Arl8b and KIF5B together control the critical step in NK cell cytotoxicity. 

KIF5B is also a critical transporter of p53 and c-Myc to the cytoplasm for degradation. However, subcellular localization of Arl8b and p53-dependent cell death was shown to occur through knockdown of acetylation subunit NatC. As a consequence, p53 is stabilized, phosphorylated and significantly activates transcription of downstream proapoptotic genes. In the absence KIF5B, or presence of  mutants p53 and c-Myc aggregate in the nucleus where they signal DNA damage-induced apoptosis through the control of p53 by endogenous c-Myc (in vivo).

Finely tuned, frequently used KIF5B in NK cells expressing GNLY may induce effects on local tissue blood pressure, as was discovered by expression of Renin-Angiotensin vasoactive proteins AT1, AT2, and ANP in pregnancy-induced uterine NK cells. Inflammation signaling, via tissue bound NK cells may result from stretch-mediated release of angiotensin II, which is coupled with p53 acetylation apoptosis and activation of p53. This may prolong upregulation of the local renin-angiotensin system, increase susceptibility of target cells to apoptosis and signal adaptive immune cells. 

Somewhere in the balance between NatC knockdown induced apoptosis and angiotensin II induced apoptosis p53 may direct traffic to keep your cells healthy!

 

An Integrated P53 Puzzle - Glycolysis in Cancer, Diabetes and Immunity!

Oxygen poor, hypoxic tissue promotes a cellular shift in mitochondrial metabolism from OXPHOS to less energy efficient glycolysis. Each shift induces environmental, epigenetic and genetic factors that alter a cells response to insult, attack and disease. Endothelial tip cells at micro-vessel ends are predominantly glycolytic. However, deletion of PFKFB3, the critical regulator of glycolysis reduced the sprouting of micro-vessel tips and elevated PFKFB3 levels improved tip cell sprouting, direction and cell behavior.

In response to DNA damage p53 promotes nucleotide biosynthesis by repressing the expression of PFKFB3. This increases the flux of glucose, through the pentose phosphate pathway (PPP) to increase nucleotide production, which results in more efficient repair of DNA damage and cell survival.

In Panc1 pancreatic cells, pro-apoptotic TGFβ1 enhanced PFKFB3 expression and stimulated glycolysis. Extracellular lactate induces endothelial mesenchymal transition (EMT) by remodeling the extracellular matrix and releasing activated TGFβ1.  TGFβ is a potent immunosuppressive cytokine that can impede development and function of natural killer (NK) and other immune cells. Furthermore, high extracellular lactate levels can contribute to immune evasion, thereby promoting tumor growth and metastasis. In tumor microenvironments glycolysis also leads to accumulated lactate, which stabilizes hypoxia inducible factor 1α (HIF-1) and upregulates the expression of anti-apoptotic, VEGF (in axis with NRP-1 dependency) resulting in angiogenesis and stimulation of cell migration. 

Hypoxia induces the loss of differentiation markers of several tumor types while increasing expression of embryonic markers such as transcription factors NANOG, OCT4, SOX2, and the Notch ligand. This reprogramming, toward a cancer stem phenotype is associated with increased tumorigenesis. In non-small cell lung carcinoma cells hypoxia increased NANOG expression that contributed to hypoxia-induced tumor cell resistance against cytotoxic lymphocyte (CTL)-mediated lysis.

Under stress the outer mitochondrial membrane incorporates Pink1, which binds and phosphorylates p53 at serine 392 and aids phagophore formation to enhance mitophagy. This reduces transport of p53-s392 to the nucleus where it would otherwise disrupt transcription of Nanog. p53 regulates Pink1 and Parkin, which regulate mitochondrial antigen presentation of both MHC classes. 

The development of type 1 diabetes involves a complex interaction between pancreatic β-cells and cells of the innate and adaptive immune systems. Analyses of the interactions between NK cells, NKT cells, dendritic cell populations and T cells have highlighted how these can influence the onset of autoimmunity. NK cells were observed in the pancreas, in NoD mice before T cell infiltration and are critically required in the pancreas for accelerated diabetes.

The islet in type 2 diabetes (T2D) is characterized by IAPP amyloid deposits, a protein co-expressed with insulin by β-cells. Human IAPP (hIAPP) misfolded protein stress activates HIF-1/PFKFB3 signaling, which increases glycolysis, mitochondrial fragmentation and perinuclear clustering, considered protective against increased cytosolic Ca2+, characteristic of amylin toxic oligomer stress. β-cells in adult humans are minimally replicative and fail to execute the second pro-regenerative phase of the HIF-1/PFKFB3 injury pathway. β-cells remain trapped in the pro-survival first phase of the HIF-1 injury repair response with a metabolism and mitochondrial network adapted to slow the rate of cell attrition at the expense of β-cell function. The senescent-like state may support the reduced NK cell activity and presence of more pro-inflammatory M1 macrophages in T2D

p53 deficient tumors can be metabolically reprogrammed and regressed by deleting isoforms of p63 or p73 to upregulate IAPP and amylin, which through the calcitonin receptor (CalcR) and receptor-activity-modifying-protein 3 (RAMP3) inhibit glycolysis, induce ROS and apoptosis. In epidermal keratinocytes p63 promotes glycolytic metabolism  by binding PFKFB3 consensus sites required for mRNA and protein expression.

Senescent cells typically upregulate anti-apoptotic pathways, and are preferentially susceptible to inhibition of these pro-survival mechanisms. This has been dubbed the ‘Achilles heel’ of senescent cells and may relate to the low mitochondrial membrane potential found in many senescent cells that ease the release of apoptosis-stimulating factors from mitochondria to promote survival. Similar weaknesses may be present through glycolysis in cancer, diabetes, other diseases and immune response.