Rhythm and Refrain: In Between Philosophy and Arts (2016)

<p>Pictorial representation of few important host defense response and apoptosis related genes that were differentially expressed (up regulated [green]; down regulated [red]) in HEV replicon transfected cell cultures compared to pcDNA3 only control.</p

Evaluation of the Dependence of the Performance of the Circuit in Figure 4 on the Feedback Strength (i.e., on the Mean Amplitude of Current Injection from the Readout Back into Neurons in the Circuit)

<p>For each feedback strength that was evaluated, the readouts were trained and tested for this feedback strength as for the preceding experiments. Error bars in (B–D) denote standard error. These control experiments show that the feedback is essential for the performance of the circuit.</p

Organization of Input and Output Streams for the Three Computational Tasks Considered in the Computer Simulations

<div><p>Each input stream consisted of multiple spike trains that provided synaptic inputs to individually chosen subsets of neurons in the recurrent circuit (which is indicated by a gray rectangle).</p><p>(A,C) Input streams consisted of multiple Poisson spike trains with a time-varying firing rate <i>r</i>_i(<i>t</i>).</p><p>(B) Input consisted of a burst (“cue”) in one spike train (which marks the beginning of a time interval) and independent Poisson spike train (“noise”) in the other input channels.</p><p>(A–C) The actual outputs of the readouts (that were trained individually for each computational task) is shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020165#pcbi-0020165-g002" target="_blank">Figures 2</a>–<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020165#pcbi-0020165-g004" target="_blank">4</a>.</p></div

Design of CRISPR-Cas9 mediated lncBISPR gene deletion using dual gRNA-Cas9 and homologous recombination (HR) donor constructs.

<p>a. Genomic organisation of BST2 (Tetherin) and LncBISPR gene on human chromosome 19. b. Schematic representing the targeting sites of gRNA1 and gRNA2 in exon 2 and exon 5 of lncBISPR gene respectively. c. Schematic representing the location of primers P1 and P3 used for PCR screening of BISPR gene deletion. d. GFP positive Huh7 cells, post dual gRNA-Cas9, HR donor construct transfection and Puromycin selection (40x objective, Bar = 20μm).</p

Computational Architectures Considered in Theorems 1 and 2

<div><p>(A) A fixed circuit <i>C</i> whose dynamics is described by the system (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020165#pcbi-0020165-e003" target="_blank">Equation 3</a>).</p><p>(B) An arbitrary given <i>n^th</i> order dynamical system (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020165#pcbi-0020165-e004" target="_blank">Equation 4</a>) with external input <i>u</i>(<i>t</i>).</p><p>(C) If the input <i>v</i>(<i>t</i>) to circuit <i>C</i> is replaced by a suitable feedback <i>K</i>(<b>x</b>(<i>t</i>),<i>u</i>(<i>t</i>)), then this fixed circuit <i>C</i> can simulate the dynamic response <i>z</i>(<i>t</i>) of the arbitrarily given system shown in B, for any input stream <i>u</i>(<i>t</i>).</p><p>(D) Arbitrary given FSM <i>A</i> with l state.</p><p>(E) A noisy fading-memory system with feedback can reliably reproduce the current state <i>A</i>(<i>t</i>) of the given FSM <i>A,</i> except for timepoints <i>t</i> shortly after <i>A</i> has switched its state.</p></div

Hepatitis E Virus (HEV) egress: Role of BST2 (Tetherin) and interferon induced long non- coding RNA (lncRNA) BISPR

<div><p>Background</p><p>The biology of Hepatitis E Virus (HEV), a common cause of epidemic and sporadic hepatitis, is still being explored. HEV exits liver through bile, a process which is essential for its natural transmission by feco-oral route. Though the process of this polarised HEV egress is not known in detail, HEV pORF3 and hepatocyte actin cytoskeleton have been shown to play a role.</p><p>Methods</p><p>Our transcriptome analysis in Hepatitis E virus (HEV) replicon transfected Huh7 cells at 24 and 72 hrs indicated that at 24hrs, both LncBISPR and BST2, expressed by a bidirectional promoter were highly upregulated whereas at 72 hrs, BST2 expression was comparatively reduced accompanied by normal levels of BISPR. These findings were confirmed by qPCR analysis. Co-localisation of BST2 and HEV pORF2 was confirmed in HEV transfected Huh7 by confocal microscopy. To investigate the role of BISPR/BST2 in HEV life cycle, particularly virus egress, we generated Huh7 cells with ~8kb deletion in BISPR gene using Crispr-Cas9 system. The deletion was confirmed by PCR screening, Sanger sequencing and Real time PCR. Virus egress in ΔBISPR Huh7 and Huh7 cells was compared by measuring HEV positive strand RNA copy numbers in cell lysates and culture supernatants at 24 and 72 hrs post HEV replicon transfection and further validated by western blot for HEV pORF2 capsid protein.</p><p>Results</p><p>ΔBISPR Huh7 cells showed ~8 fold increase in virus egress at 24 hrs compared to Huh7 cells. No significant difference in virus egress was observed at 72hrs. Immunohistochemistry in histologically normal liver and HEV associated acute liver failure revealed BST2 overexpression in HEV infected hepatocytes and a dominant canalicular BST2 distribution in normal liver in addition to the cytoplasmic localisation reported in literature.</p><p>Conclusions</p><p>These findings lead us to believe that BISPR and BST2 may regulate egress of HEV virions into bile <i>in vivo</i>. This system may also be used to scale up virus production <i>in vitro</i>.</p></div

Representative images showing immunohistochemical expression of BST2 (Tetherin) and HEV pORF2 <i>in vivo</i>.

<p>Expression of BST2 (Tetherin) in histologically normal liver [a-d (inset in ‘a’ represents subcapsular region)], lymph node (f) and spleen (inset f). Blood vessels in liver (b) served as positive internal control for BST2 (Tetherin) staining. Hepatocytes show cytoplasmic (c) and canalicular staining (arrows in Fig d and inset Fig d). Figs ‘e’ (liver) and ‘h’ (spleen) represent negative IHC controls with secondary antibody only. Fig ‘g’ represents negative control for pORF2 staining in histologically normal liver. Figs ‘i-t’ depict immediate serial sections of HEV associated Acute liver failure tissue biopsy (ALF) stained with Hematoxylin and Eosin (H&E; i-l), anti-HEV pORF2 IHC (m-p) and anti-BST2 (Tetherin) IHC (q-t). Similar cell populations stained with both the antibodies (Arrows in Figs l, p and t). Inset in Fig ‘s’ depicts perinuclear staining of BST2 (Tetherin) in HEV associated ALF case. Images ‘i, m, q’ were taken with 4x objective (Bar = 0.5mm); ‘a, h, g’ with 10x objective (Bar = 0.25mm); ‘b, c, d, e, f, g, j, k, n, o, r, s’ with 20x objective (Bar = 0.1mm) and ‘l, p, t, inset d and inset s’ with 40x objective (Bar = 50μm).</p

Analysis of HEV virion egress from ΔBISPR Huh7 cells.

<p>a. Bar diagram showing expression of lncBISPR and BST2 (Tetherin) in HEV replicon transfected ΔBISPR Huh7 (test) 24 hrs post transfection and untransfected Huh7 cells (control) (n = 3; error bars represent standard deviation, *p-value <0.01). GAPDH was used as a reference gene for normalization. b. Confocal images (40x objective) representing staining patterns of HEV pORF2 and BST2 (Tetherin) in untransfected and HEV transfected ΔBISPR Huh7 cells, 24hrs post transfection. Bars represent 20μm. c. Bar diagram representing percentage of HEV RNA in culture supernatants of HEV transfected ΔBISPR Huh7 and Huh7 cells at 24 hrs post transfection (n = 3; error bars represent standard deviation). d. Ponceau-S and anti-HEV pORF2 stained western blot analysis of HEV virions in the culture supernatants of HEV transfected Huh7 (lane 6) and ΔBISPR Huh7 cells (lanes 2–4) 24hrs post transfection. Capsid protein pORF2 is shown by arrow heads. Lane 5 represents the negative control sample (culture supernatant from untransfected Huh7 cells). Lane 1 represents prestained protein molecular weight marker (Puregene, United States).</p

BST2 (Tetherin) and BISPR expression in HEV transfected Huh7 cells.

<p>a. Bar chart representing BST2 (Tetherin) RNA levels in untransfected (black), wild type HEV replicon (red) and replication deficient HEV RNA (blue) transfected Huh7 cells at 12, 24 and 72 hrs post transfection. Relative expression values have been normalised with respect to GAPDH gene (n = 3; error bars represent standard deviation, *p value <0.01). b. Bar chart representing BISPR RNA levels in untransfected (black), wild type HEV replicon (red) and replication deficient HEV RNA (blue) transfected Huh7 cells at 12, 24 and 72 hrs post transfection. Relative expression values have been normalised with respect to GAPDH gene (n = 3; error bars represent standard deviation, *p value <0.01). c. Confocal images (40x objective) representing dual Immunofluorescence colocalisation patterns of HEV pORF2 and BST2 (Tetherin) in HEV transfected Huh7 cells in replicate samples (panels 1, 2: cytoplasmic and membranous, 3: perinuclear), 24hrs post transfection. Panels 4 represents staining pattern of untransfected Huh7 cells. Bars in panels 1–3 and 4 represent 20μm and 10μm respectively.</p

Emulation of an FSM by a Noisy Fading-Memory System with Feedback According to Theorem 5

<div><p>(A) Underlying open-loop system with noisy pattern detectors ₁, …, <i>_k</i> and suitable fading-memory readouts <i>Ĥ</i>₁, …, <i>Ĥ_l</i> (which may also be subject to noise).</p><p>(B) Resulting noise-robust emulation of an arbitrary given FSM by adding feedback to the system in (A). The same readouts as in (A) (denoted <i>CL</i> − <i>Ĥ_j</i>(<i>t</i>) in the closed loop) now encode the current state of the simulated FSM.</p></div