Biology subjects

Roy, A.

Publications and source records attributed to Roy, A..

At least 163 records · Page 9Linked to original sources

bioRxiv2021

Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalks between SA-dependent immunity and phosphate-starvation responses

The propensity for polyphosphorylation makes myo-inositol derivatives, the inositol polyphosphates (InsPs), especially phytic acid or inositol hexakisphosphate (InsP6) the major form of phosphate storage in plants. Acts of pyrophosphorylation on InsP6 generates InsP7 or InsP8 containing high-energy phosphoanhydride bonds that are harnessed during energy requirements of a cell. Also implicated as co-factors for several phytohormone signaling networks, InsP7/InsP8 modulate key developmental processes. With recent identification as the common moeity for transducing both jasmonic acid (JA) and phosphate-starvation responses (PSR), InsP8 is the classic example of a metabolite that may moonlight crosstalks to different cellular pathways during diverse stress adaptations. We show here that Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE (IPK1), INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), and DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASE 2 (VIH2), but not other InsP-kinases, suppress basal salicylic acid (SA)-dependent immunity. In ipk1, itpk1 or vih2 mutants, elevated endogenous SA levels and constitutive activation of defense signaling lead to enhanced resistance against the virulent Pseudomonas syringae pv tomato DC3000 (PstDC3000) strain. Our data reveal that activated SA-signaling sectors in these mutants modulate expression amplitudes of phosphate-starvation inducible (PSI)-genes, reported earlier. In turn, via mutualism the heightened basal defenses in these mutants require upregulated PSI-gene expressions likely highlighting the increased demand of phosphates required to support immunity. We demonstrate that SA is induced in phosphate-deprived plants, however its defense-promoting functions are likely diverted to PSR-supportive roles. Overall, our investigations reveal selective InsPs as crosstalk mediators among diverse signaling networks programming stress-appropriate adaptations.

plant biology↗

bioRxiv2021

Simple and Rapid High-Throughput Assay to Identify HSV-1 ICP0 Transactivation Inhibitors

Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that results in lifelong infections due to its ability to cycle between lytic replication and latency. As an obligate intracellular pathogen, HSV-1 exploits host cellular factors to replicate and aid in its life cycle. HSV-1 expresses infected cell protein 0 (ICP0), an immediate-early regulator, to stimulate the transcription of all classes of viral genes via its E3 ubiquitin ligase activity. Mechanisms by which ICP0 activates viral gene expression and the cellular factors involved are largely unknown. Here we report an automated, inexpensive, and rapid high-throughput approach to examine the effects of small molecule compounds on ICP0 transactivator function in cells. Two HSV-1 reporter viruses, KOS6{beta} (wt) and dlx3.1-6{beta} (ICP0-null mutant), were used to monitor ICP0 transactivation activity through the HSV-1 ICP6 promoter::lacz expression cassette. A [≥]10-fold difference in {beta}-galactosidase activity was observed in cells infected with KOS6{beta} compared to dlx3.1-6{beta}, demonstrating that ICP0 potently transactivates the ICP6 promoter. We established the robustness and reproducibility with a Z'- factor score of [≥]0.69, an important criterium for high-throughput analyses. Approximately 19,000 structurally diverse compounds were screened and 76 potential inhibitors of the HSV-1 transactivator ICP0 were identified. We expect this assay will aid in the discovery of novel inhibitors and tools against HSV-1 ICP0. Using well-annotated compounds could identify potential novel factors and pathways that interact with ICP0 to promote HSV-1 gene expression.

microbiology↗

bioRxiv2020

A highly contiguous genome assembly of a major forest pest, the Eurasian spruce bark beetle Ips typographus

The Eurasian spruce bark beetle (Ips typographus [L.]), is a major killer of spruce forests across the Palearctic. During epidemics, it can destroy over 100 million cubic meters of spruce trees in a single year. Here we report a 236 Mb, highly contiguous I. typographus genome assembly using PacBio long-read sequencing. The final phased assembly had a contig N50 of 6.65 Mb in 272 contigs and was predicted to contain 23,923 protein-coding genes. Comparative genomic analysis revealed expanded gene families associated with plant cell wall degradation, including pectinases, aspartyl proteases, and glycosyl hydrolases. In todays forests, increasingly stressed by global warming, this resource can assist in mitigating bark beetle outbreaks by developing novel pest control strategies. Further, this first whole-genome sequence from the genus Ips provides timely resources to address important questions about the evolutionary biology and ecology of Curculionidae, the true weevils, one of the largest animal families.

genomics↗

bioRxiv2020

A pipeline to characterize local cortical folds by mapping them to human-interpretable shapes

BackgroundVariations in regional cortical folds across individuals have been examined using computationally-derived morphological measures, or by manual characterization procedures that map distinct variants of a regional fold to a set of human-interpretable shapes. Although manual mapping approaches have proven useful for identifying morphological differences of clinical relevance, such procedures are subjective and not amenable to scaling. New MethodWe propose a 3-step pipeline to develop computational models of manual mapping. The steps are: represent regional folds as feature vectors, manually map each feature vector to a shape-variant that the underlying fold represents, and train classifiers to learn the mapping. ResultsFor demonstration, we chose a 2D-problem of detecting within slice discontinuity of medial and lateral sulci of orbitofrontal cortex (OFC); the discontinuity may be visualized as a broken H-shaped pattern, and is fundamental to OFC-type-characterization. The classifiers predicted discontinuities with 86-95% test-accuracy. Comparison with Existing MethodsThere is no existing pipeline that automates a manual characterization process. For the current demonstration problem, we conduct multiple analyses using existing softwares to explain our design decisions, and present guidelines for using the pipeline to examine other regional folds using conventional or non-conventional morphometric measures. ConclusionWe show that this pipeline can be useful for determining axial-slice discontinuity of sulci in the OFC and can learn structural-features that human-raters may rely on during manual-characterization.The pipeline can be used for examining other regional folds and may facilitate discovery of various statistically-reliable 2D or 3D human-interpretable shapes that are embedded throughout the brain.

neuroscience↗

bioRxiv2020

Intranasal Administration of ACIS KEPTIDE™ Prevents SARS-CoV2-Induced Acute Toxicity in K18-hACE2 Humanized Mouse Model of COVID-19: A Mechanistic Insight for the Prophylactic Role of KEPTIDE™ in COVID-19

Previously, we have demonstrated that ACIS KEPTIDE, a chemically modified peptide, selectively binds to ACE-2 receptor and prevents the entry of SARS-CoV2 virions in vitro in primate kidney Cells. However, it is not known if ACIS KEPTIDE attenuates the entry of SARS-CoV2 virus in vivo in lung and kidney tissues, protects health, and prevent death once applied through intranasal route. In our current manuscript, we demonstrated that the intranasal administration of SARS-CoV2 (1*106) strongly induced the expression of ACE-2, promoted the entry of virions into the lung and kidney cells, caused acute histopathological toxicities, and mortality (28%). Interestingly, thirty-minutes of pre-treatment with 50 g/Kg Body weight ACIS normalized the expression of ACE-2 via receptor internalization, strongly mitigated that viral entry, and prevented mortality suggesting its prospect as a prophylactic therapy in the treatment of COVID-19. On the contrary, the peptide backbone of ACIS was unable to normalize the expression of ACE-2, failed to improve the health vital signs and histopathological abnormalities. In summary, our results suggest that ACIS is a potential vaccine-alternative, prophylactic agent that prevents entry of SARS-CoV2 in vivo, significantly improves respiratory health and also dramatically prevents acute mortality in K18-hACE2 humanized mice. HighlightsO_LIACIS KEPTIDE stimulates the internalization of ACE-2 receptor (Fig. 2) and buffers the membrane localization of ACE-2 receptors (Fig. 2, 6 & 8). Intranasal inoculation of SARS-CoV2 upregulates the expression of ACE-2 in lung epithelium (Fig.6) and kidney tubular cells (Fig.8). ACIS KEPTIDE normalizes the expression of ACE-2 in the kidney tubular cells of virus-treated K18-hACE2mice (Fig. 8). C_LIO_LIACIS KEPTIDE completely prevents the entry of SARS-CoV2 in Bronchiolar epithelium (Fig.6), alveolar parenchyma (Fig. 6), and kidney tubular cells (Fig.8). C_LIO_LIACIS KEPTIDE improves the pulmonary (Fig. 5) and renal pathological changes (Fig. 7) caused by the SARS-CoV2 virus insult. C_LIO_LIIntranasal administration of 0.05% Beta-propiolactone ({beta}PL)-inactivated SARS-CoV2 (1 *106) causes significant death (28%) in K18-hACE2 humanized mice after 24 hrs of intranasal inoculation (Supplemental videos) suggesting that SARS-CoV2 does not require its infective properties and genetic mechanism to be functional to cause mortality. C_LIO_LIThe peptide backbone of ACIS KEPTIDE provides much less and insignificant protection in the prevention of pathological changes in Lungs (Fig.5 & 6) and Kidney (Fig.7 & 8). Peptide failed to normalize the upscaled expression of ACE-2 in kidney tubular cells (Fig.8) of SARS-CoV2-treated K18-hACE2 mice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/378257v1_fig2.gif" ALT="Figure 2"> View larger version (51K): org.highwire.dtl.DTLVardef@15c9911org.highwire.dtl.DTLVardef@453819org.highwire.dtl.DTLVardef@65f8a0org.highwire.dtl.DTLVardef@a602d8_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 2.C_FLOATNO Effect of KEPTIDE on the Expression of ACE-2 Receptor on the Membrane of CALU-3 Human Lung Cells. Human lung epithelial cells. CALU-3 cells were grown in complete DMEM cells for 2 days until it reached 70% confluency followed by starving with serum for 2 hrs. After that, 25 M of ACIS KEPTIDE were treated for 30 mins, 1 hr, 2 hrs and 6 hrs. After each time point cells were fixed and stained for ACE-2 (Green; Rabbit anti-ACE-2 antibody; Abcam; 1:250 dilution) and KEPTIDE (blue). Thirty minutes of KEPTIDE treatment significantly stimulated the internalization of ACE-2 along with KEPTIDE. Subsequent incubation periods displayed significant down-regulation of ACE-2 receptors. Experiments were confirmed after three different experiments. C_FIG O_FIG O_LINKSMALLFIG WIDTH=154 HEIGHT=200 SRC="FIGDIR/small/378257v1_fig6.gif" ALT="Figure 6"> View larger version (98K): org.highwire.dtl.DTLVardef@104bd7forg.highwire.dtl.DTLVardef@34ecc1org.highwire.dtl.DTLVardef@a3ac74org.highwire.dtl.DTLVardef@18f0570_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 6.C_FLOATNO Protective effect of ACIS KEPTIDE on the expression of ACE-2 and the entry of SARS-CoV2 virions in lung of SARS-CoV2-insulted K18-hACE2 mice. (A-D) Dual IHC staining of ACE-2 (red) and SARS-CoV2 (brown) in bronchiolar epithelium in (A) vehicle-treated (0.05% PL-inactivated VEROE6 sup, (B)virus, (C) virus + KEPTIDE, and (D) virus + peptide-treated K18-hACE2 mice (n= 7-8). (a-h) Magnified views of outer layers and inner layers of bronchiolar epithelia of respective images enclosed in a dotted squares. Arrows were justified in the bottom of each image. Results are confirmed after three independent experiments. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/378257v1_fig8.gif" ALT="Figure 8"> View larger version (87K): org.highwire.dtl.DTLVardef@ae105aorg.highwire.dtl.DTLVardef@1b39472org.highwire.dtl.DTLVardef@d6e3e9org.highwire.dtl.DTLVardef@ce6d6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 8.C_FLOATNO Protective effect of ACIS KEPTIDE on the expression of ACE-2 and prevention of SARS-CoV2 entry in kidney of K18-hACE2 mice twenty-four hours post SARS-CoV2 inoculation. Eight to ten weeks old K18-hACE2 mice (n=7-8 per group) were intranasally administered with 50 g/kg Bwt KEPTIDE or 50 g/kg Bwt peptide for 30 mins followed by inoculation with PL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% PL-treated VEROE6 sup); in group 2, mice (n=7) were treated with virus only (0.05% PL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 4 mice (n=8) were treated with virus + PEPTIDE. (Results are confirmed after three independent experiments. (A-D) Dual IHC of ACE-2 (red) and SARS-CoV2 (brown) in tubular epithelium in (A) vehicle, (B) Virus, (C) Virus + KEPTIDE-, and (D) Virus + peptide-treated groups. (a) showed magnified view of kidney cortex vehicle-treated mouse with basal expression of ACE-2 (red), (b) magnified view of kidney of virus-tread animal. Upscaled expression of ACE-2(red) with degenerated Bowmans capsule and invasion of SARS CoV2 (Brown arrow). (c) Magnified view of Glomerulus of Virus + KEPTIDE-treated group. Significantly less ACE-2 expression (red arrow) and no virus-infiltration were noted. (d) Elevated expression of ACE-2 in virus +Peptide-treated group. Results were confirmed after three different experiments in 7-8 animals. C_FIG O_FIG O_LINKSMALLFIG WIDTH=190 HEIGHT=200 SRC="FIGDIR/small/378257v1_fig5.gif" ALT="Figure 5"> View larger version (123K): org.highwire.dtl.DTLVardef@1072b3aorg.highwire.dtl.DTLVardef@1a70702org.highwire.dtl.DTLVardef@bd227corg.highwire.dtl.DTLVardef@12335bd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 5.C_FLOATNO ACIS KEPTIDE protects acute histopathological changes in Lungs of K18-hACE2 mice twenty-four hours post SARS-CoV2 inoculation. Eight to ten weeks old K18-hACE2 mice (n=7-8 per group) were intranasally administered with 50 g/kg Bwt KEPTIDE or 50 g /kg Bwt peptide for 30 mins followed by inoculation with PL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% PL-treated media); in group 2, mice (n=7 were treated with virus only (0.05% PL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 4 mice (n=8) were treated with virus + PEPTIDE. (A-D) Hematoxylin Background staining of small airway alveolar parenchyma. (E-H) H & E staining of Bronchiolar epithelium, surrounding cartilaginous and alveolar parenchyma. Magnified views of bronchiolar epithelia of (Ei) Control (orange arrow indicates intact epithelial lining: orange star demonstrates preserved connective tissue), (Fii) Virus only (thin red arrow indicates the degenerated bronchiolar epithelium), (Giii) Virus + KEPTIDE (thin blue arrow indicates protected epithelium), and (Hiv) Virus + Peptide-treated (thin green arrow indicates degenerated epithelium). Results are confirmed after three different experiments in 7-8 animals. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/378257v1_fig7.gif" ALT="Figure 7"> View larger version (150K): org.highwire.dtl.DTLVardef@9d3c17org.highwire.dtl.DTLVardef@d40a51org.highwire.dtl.DTLVardef@f17652org.highwire.dtl.DTLVardef@8f3230_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 7.C_FLOATNO Effect of ACIS KEPTIDE on the protection of acute histopathological changes in Kidney of K18-hACE2 mice twenty-four hours post SARS-CoV2 inoculation. Eight to ten weeks old K18-hACE2 mice (n=7-8 per group) were intranasally administered with 50 g/kg Bwt KEPTIDE or 50 g/kg Bwt peptide for 30 mins followed by inoculation with PL-inactivated 1*106 virus. In group1 mice (n=8) were inoculated with vehicle only (0.05% PL-treated VEROE6 sup); in group 2, mice (n=7) were treated with virus only (0.05% PL-inactivated; in group 3, mice (n=8) were treated with virus +KEPTIDE; and, in group 4 mice (n=8) were treated with virus + PEPTIDE. (A-D) H & E staining of kidney cortex with detailed structures of glomeruli, tubular epithelium, and Bowmans capsule in all 4 groups. Results are confirmed after three independent experiments. C_FIG

animal behavior and cognition↗

bioRxiv2020

A novel human fetal liver-derived model reveals that MLL-AF4 drives a distinct fetal gene expression program in infant ALL

Although 90% of children with acute lymphoblastic leukemia (ALL) are now cured1, the prognosis of infant-ALL (diagnosis within the first year of life) remains dismal2. Infant-ALL is usually caused by a single genetic hit that arises in utero: rearrangement of the MLL/KMT2A gene (MLL-r). This is sufficient to give rise to a uniquely aggressive and treatment-refractory leukemia compared to older children with the same MLL-r3-5. The reasons for disparate outcomes in patients of different ages with identical driver mutations are unknown. This paper addresses the hypothesis that fetal-specific gene expression programs co-operate with MLL-AF4 to initiate and maintain infant-ALL. Using direct comparison of fetal and adult HSC and progenitor transcriptomes we identify fetal-specific gene expression programs in primary human cells. We show that MLL-AF4-driven infant-ALL, but not MLL-AF4 childhood-ALL, displays expression of fetal-specific genes. In a direct test of this observation, we find that CRISPR-Cas9 gene editing of primary human fetal liver cells to produce a t(4;11)/MLL-AF4 translocation replicates the clinical features of infant-ALL and drives infant-ALL-specific and fetal-specific gene expression programs. These data strongly support the hypothesis that fetal-specific gene expression programs co-operate with MLL-AF4 to initiate and maintain the distinct biology of infant-ALL.

cancer biology↗

bioRxiv2020

Autocrine Signaling by Receptor Tyrosine Kinases in Urothelial Carcinoma of the Bladder

Comprehensive characterizations of bladder cancer (BCa) have established molecular phenotype classes with distinct alterations and survival trends. Extending these studies within the tyrosine kinase (TK) family to identify disease drivers could improve our use of TK inhibitors to treat specific patient groups or individuals. We examined the expression distribution of TKs as a class (n = 89) in The Cancer Genome Atlas (TCGA) muscle invasive BCa data set (n >400). Patient profiles of potentially oncogenic alterations (overexpression and/or amplification) clustered TKs into 3 groups; alterations of group 1 and 3 TKs were associated with significantly worse patient survival relative to those without alterations. Many TK pathways induce epithelial-to-mesenchymal transition (EMT), which promotes tumor invasiveness and metastasis. Overexpression and/or amplification among 9 EMT transcriptional activators occurred in 43% of TCGA cases. Co-occurring alterations of TKs and EMT transcriptional activators involved most group 1 TKs; 24% of these events were associated with significantly worse patient survival. Co-occurring alterations of receptor TKs and their cognate ligands occurred in 16% of TCGA cases and several BCa-derived cell lines. Suppression of GAS6, MST1 or CSF1, or their respective receptors (AXL, MST1R and CSF1R), in BCa cell lines was associated with decreased receptor activation, cell migration, cell proliferation and anchorage independent cell growth. These studies reveal the patterns and prevalence of potentially oncogenic TK pathway-related alterations in BCa and identify specific alterations associated with reduced BCa patient survival. Detection of these features in BCa patients could better inform TK inhibitor use and improve clinical outcomes.

cancer biology↗

bioRxiv2020

ACIS, A Novel KepTide™, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19

Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein "spike" (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally.

biochemistry↗

bioRxiv2020

Spatial information transfer in hippocampal place cells depends on trial-to-trial variability, symmetry of place-field firing and biophysical heterogeneities

The relationship between the feature-tuning curve and information transfer profile of individual neurons provides vital insights about neural encoding. However, the relationship between the spatial tuning curve and spatial information transfer of hippocampal place cells remains unexplored. Here, employing a stochastic search procedure spanning thousands of models, we arrived at 127 conductance-based place-cell models that exhibited signature electrophysiological characteristics and sharp spatial tuning, with parametric values that exhibited neither clustering nor strong pairwise correlations. We introduced trial-to-trial variability in responses and computed model tuning curves and information transfer profiles, using stimulus-specific (SSI) and mutual (MI) information metrics, across locations within the place field. We found spatial information transfer to be heterogeneous across models, but to reduce consistently with increasing degrees of variability. Importantly, whereas reliable low-variability responses implied that maximal information transfer occurred at high-slope regions of the tuning curve, increase in variability resulted in maximal transfer occurring at the peak-firing location in a subset of models. Moreover, experience-dependent asymmetry in place-field firing introduced asymmetries in the information transfer computed through MI, but not SSI, and the impact of activity-dependent variability on information transfer was minimal compared to activity-independent variability. Biophysically, we unveiled a many-to-one relationship between different ion channels and information transfer, and demonstrated critical roles for N-methyl-D-aspartate receptors, transient potassium and dendritic sodium channels in regulating information transfer. Our results emphasize the need to account for trial-to-trial variability, tuning-curve shape and biological heterogeneities while assessing information transfer, and demonstrate ion-channel degeneracy in the regulation of spatial information transfer.

neuroscience↗

bioRxiv2020

Improving laser standards for three-photon microscopy

SignificanceThree-photon excitation microscopy has double-to-triple the penetration depth in biological tissue over two-photon imaging and thus has the potential to revolutionize the visualization of biological processes in vivo. However, unlike the plug-and-play operation and performance of lasers used in two-photon imaging, three-photon microscopy presents new technological challenges that require a closer look at the fidelity of laser pulses. AimWe implemented state-of-the-art pulse measurements and developed new techniques for examining the performance of lasers used in three-photon microscopy. We then demonstrated how these techniques can be used to provide precise measurements of pulse shape, pulse energy and pulse-to-pulse intensity variability, all of which ultimately impact imaging. ApproachWe built inexpensive tools, e.g., a second harmonic generation frequency resolved optical gating (SHG-FROG) device, and a deep-memory diode imaging (DMDI) apparatus, to examine laser pulse fidelity. ResultsFirst, SHG-FROG revealed very large third order dispersion (TOD). This extent of phase distortion prevents the efficient temporal compression of laser pulses to their theoretical limit. Furthermore, TOD cannot be quantified when using a conventional method of obtaining the laser pulse duration, e.g., when using an autocorrelator. Finally, DMDI showed the effectiveness of detecting pulse-to-pulse intensity fluctuations on timescales relevant to three-photon imaging, which were otherwise not captured using conventional instruments and statistics. ConclusionsThe distortion of individual laser pulses caused by TOD poses significant challenges to three-photon imaging by preventing effective compression of laser pulses and decreasing the efficiency of nonlinear excitation. Moreover, an acceptably low pulse-to-pulse amplitude variability should not be assumed. Particularly for low repetition rate laser sources used in three-photon microscopy, pulse-to-pulse variability also degrades image quality. If three-photon imaging is to become mainstream, our diagnostics may be used by laser manufacturers to improve system design and by end-users to validate the performance of their current and future imaging systems.

bioengineering↗

bioRxiv2020

Three-dimensionally preserved 'Stage IIIb' fossil down feather supports developmental modularity in feather evolution

We describe a unique three-dimensionally preserved fossil down feather from the Late Cretaceous of Myanmar. Its morphology is highly congruent with Stage IIIb of the widely accepted Prum and Brush model of feather evolution-development. This makes the new specimen the first evidence of this developmental stage in the fossil record. The Stage IIIb diagnosis is robustly supported by the absence of a central rachis and by its paired barbules emanating from radially positioned barbs that are attached to a short calamus. Prum and Brushs model hypothesises a bifurcation in the evolution-development pathway at Stage III. Stage IIIa involves rachis development and branching into barbs. Stage IIIb involves branching of the barbs from the calamus and then further branching of the barbules from the barbs. These two pathways then converge into Stage IIIa+b where feathers produce a rachis, barbs and barbules in nested order, finally leading to Stage IV. Evolution-development studies on the morphogenesis of feathers have unequivocally shown that such feather branching can be controlled by BMP, Noggin, Shh and several other proteins. Therefore, molecular crosstalk can convert a barb into a rachis and vice versa. The topology of this down feather, consistent with specific patterns of modular protein-protein signalling already observed, provides the first definitive evidence that such signalling was responsible for the evolution of a diverse inventory of feather morphologies in non-avialan dinosaurs and early birds since the middle Jurassic.

paleontology↗

bioRxiv2020

The Effect of Refractive Blur in the Vividness of Mental Imagery

BackgroundVisual mental imagery or "seeing with the minds eye" is an everyday phenomenon. Visual mental imagery and visual perception share common neural-networks. Hence deficits that affect the visual perception may also affect visual mental imagery. AimWe aimed to study the effect of refractive blur on the vividness of mental imagery. MethodsSubjects were recruited from volunteers and divided into two groups; individuals with refractive errors-Ametropes(AM), and individuals without refractive errors - Emmetropes(EM). After filling in the Verbalizer-Visualizer-Questionnaire (VVQ), the subjects were asked to perform a mental imagery task with and without refractive blur. The participants were asked to generate a mental image of a specific object initially with eyes closed, eyes open and then with refractive blur in random order, and then judge the vividness of the mental image on a Likert scale ranging from 1 (low vividness) to 5 (good vividness). The EM participants had to wear a + 2D spectacles to produce refractive blur. ResultsA total of 162 participants were recruited to the study. Of these 73 were EM and 89 were AM. Of the AM, 30 had additional astigmatism. The mean VVQ score was 64.9(11.2). The mean refractive error was 1.8(1.3)D. Following the mental imagery task, at baseline with eyes closed, 138 (85.5%)subjects had vivid mental imagery close to visual perception(Likert scale:5). With the opening of the eyes, the vividness dropped by at least 1 point in the Likert scale in 139(85.8%). With the introduction of refractive blur, 153(94.4%) subjects had a drop in the vividness of the image by at least 1 point and 22(13.6%) subjects by at least 2 points. ConclusionIntroduction of refractory blur results in the reduction of the vividness of mental imagery.

neuroscience↗

bioRxiv2020

Phenotypic analysis of an MLL-AF4 gene regulatory network reveals indirect CASP9 repression as a mode of inducing apoptosis resistance.

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than studying single TF binding events in isolation. Chromosomal translocations of the Mixed Lineage Leukemia gene (MLL) produce MLL fusion proteins such as MLL-AF4, causing poor prognosis acute lymphoblastic leukemias (ALLs). MLL-AF4 is thought to drive leukemogenesis by directly binding to genes and inducing aberrant overexpression of key gene targets, including anti-apoptotic factors such as BCL-2. However, this model minimizes the potential for circuit generated regulatory outputs, including gene repression. To better understand the MLL-AF4 driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors, including RUNX1, that regulate target genes using feed-forward loop and cascade motifs. We used CRISPR screening in the presence of the BCL-2 inhibitor venetoclax to identify functional impacts on apoptosis. This identified an MLL-AF4:RUNX1 cascade that represses CASP9, perturbation of which disrupts venetoclax induced apoptosis. This illustrates how our GRN can be used to better understand potential mechanisms of drug resistance acquisition. Graphical abstract caption O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/179796v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@cca8f2org.highwire.dtl.DTLVardef@67b289org.highwire.dtl.DTLVardef@1fa44aeorg.highwire.dtl.DTLVardef@1b32487_HPS_FORMAT_FIGEXP M_FIG A network model of the MLL-AF4 regulatory landscape identifies feed-forward loop and cascade motifs. Functional screening using CRISPR and venetoclax identified an MLL-AF4:RUNX1:CASP9 repressive cascade that impairs drug-induced cell death. C_FIG

genomics↗

bioRxiv2020

Three-photon imaging of synthetic dyes in deep layers of the neocortex

Multiphoton microscopy has emerged as the primary imaging tool for studying the structural and functional dynamics of neural circuits in brain tissue, which is highly scattering to light. Recently, three-photon microscopy has enabled high-resolution fluorescence imaging of neurons in deeper brain areas that lie beyond the reach of conventional two-photon microscopy, which is typically limited to ~450 m. Three-photon imaging of neuronal calcium signals, through the genetically-encoded calcium indicator GCaMP6, has been used to successfully record neuronal activity in deeper neocortical layers and parts of the hippocampus. Bulk-loading cells in deeper cortical layers with synthetic calcium indicators could provide an alternative strategy for labelling that obviates dependence on viral tropism and promoter penetration. Here we report a strategy for visualized injection of a calcium dye, Oregon Green BAPTA-1 AM (OGB-1 AM), at 500-600 m below the surface of the mouse visual cortex in vivo. We demonstrate successful OGB-1 AM loading of cells in cortical layers 5-6 and subsequent three-photon imaging of orientation- and direction-selective visual responses from these cells.

neuroscience↗

bioRxiv2020

SeqEnhDL: sequence-based classification of cell type-specific enhancers using deep learning models

We propose SeqEnhDL, a deep learning framework for classifying cell type-specific enhancers based on sequence features. DNA sequences of "strong enhancer" chromatin states in nine cell types from the ENCODE project were retrieved to build and test enhancer classifiers. For any DNA sequence, sequential k-mer (k=5, 7, 9 and 11) fold changes relative to randomly selected non-coding sequences were used as features for deep learning models. Three deep learning models were implemented, including multi-layer perceptron (MLP), Convolutional Neural Network (CNN) and Recurrent Neural Network (RNN). All models in SeqEnhDL outperform state-of-the-art enhancer classifiers including gkm-SVM and DanQ, with regard to distinguishing cell type-specific enhancers from randomly selected non-coding sequences. Moreover, SeqEnhDL is able to directly discriminate enhancers from different cell types, which has not been achieved by other enhancer classifiers. Our analysis suggests that both enhancers and their tissue-specificity can be accurately identified according to their sequence features. SeqEnhDL is publicly available at https://github.com/wyp1125/SeqEnhDL.

bioinformatics↗

bioRxiv2020

The SARS-CoV-2 conserved macrodomain is a highly efficient ADP-ribosylhydrolase enzyme

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low M affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. IMPORTANCESARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 900 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

biochemistry↗

bioRxiv2019

Analysing the safe and just operating space of agriculture in the world: past, present and future

Agriculture, along with industry and household sector are three major sectors of human consumption. Agriculture has proved to be a major contributor to exceeding planetary boundaries. Here, we have explored the impact of agriculture in the Earth system processes, through eight dimensions of planetary boundaries or safe operating spaces: climate change (10.73%), freshwater use (91.56%), arable land use (37.27%), nitrogen use (95.77%), phosphorus use (87.28%), ecological footprint (19.42%), atmospheric pollution (2.52% - 38.08%) and novel entities. In this work, we have also shown role of agriculture to the socio-economic development dimensions: gender equality, employment and economic growth. We have shown that the safe operating limits for agriculture are going to decline by almost 55% (climate change), 300% (freshwater use), 50-55% (arable land use), 180% (nitrogen use), 265% (phosphorus use) and 20% (ecological footprint) in 2050, if the most inefficient way of consumption is chosen and continued. To alleviate the role of agriculture in transgressing planetary boundaries, it is indispensable to comprehend how many roles of agriculture is playing and where which target should be set to framework the national agricultural policies in coherence with attaining sustainable development goals of UN by 2030.

ecology↗

bioRxiv2019

HYPK scaffolds the Nedd8 and LC3 proteins to initiate the formation of autophagosome around the poly-neddylated huntingtin exon1 aggregates

Selective degradation of protein aggregates by autophagy is an essential homeostatic process of safeguarding cells from the effects of proteotoxicity. Among the ubiquitin-like modifier proteins, Nedd8 conjugation to misfolded proteins is prominent in stress-induced protein aggregates, albeit the function of neddylation in autophagy is unclear. Here, we report that polyneddylation functions as a post-translational modification for autophagic degradation of proteotoxic-stress induced protein aggregates. We also show that HYPK functions as an autophagy receptor in the polyneddylation-dependent aggrephagy. The scaffolding function of HYPK is facilitated by its C-terminal ubiquitin-associated domain and N-terminal tyrosine-type LC3 interacting region which bind to Nedd8 and LC3 respectively. Both Nedd8 and HYPK are positive modulators of basal and induced-autophagy, leading to desensitizing cells from protein aggregates, such as aggregates of mutant huntingtin-exon1. Thus, we propose an additive role of neddylation and HYPK in clearance of protein aggregates by autophagy, resulting in cytoprotective effect during proteotoxic stress.

cell biology↗