Cell Spike Research Articles

Ulex Europaeus Agglutinin-I-Based Magnetic Isolation for the Efficient and Specific Capture of SW480 Circulating Colorectal Tumor Cells.

The efficient and specific capture of circulating tumor cells (CTCs) from patients’ peripheral blood is of significant value in precise cancer diagnosis and cancer therapy. As fine targeting molecules, lectins can recognize cancer cells specifically due to the abnormal glycosylation of molecules on the cancer cell membrane and the specific binding of lectin with glycoconjugates. Herein, a Ulex europaeus agglutinin-I (UEA-I)-based magnetic isolation strategy was developed to efficiently and specifically capture α-1,2-fucose overexpression CTCs from colorectal cancer (CRC) patients’ peripheral blood. Using UEA-I-modified Fe3O4 magnetic beads (termed MB-UEA-I), up to 94 and 89% of target cells (i.e., SW480 CRC cells) were captured from the cell spiking complete cell culture medium and whole blood, respectively. More than 90% of captured cells show good viability and proliferation ability without detaching from MB-UEA-I. In combination with three-color immunocytochemistry (ICC) identification, MB-UEA-I has been successfully used to capture CTCs from CRC patients’ peripheral blood. The experimental results indicate a correlation between CTC characterization and tumor metastasis. Specifically, MB-UEA-I can be applied to screen early CRC by capturing CTCs when served as a liquid biopsy. The presented work offers a new insight into developing cost-effective lectin-functionalized methods for biomedical applications.

Significance of chlorine-dioxide-based oral rinses in preventing SARS-CoV-2 cell entry.

ObjectiveThis work aims to determine the efficacy of preprocedural oral rinsing with chlorine dioxide solutions to minimize the risk of coronavirus disease 2019 (COVID‐19) transmission during high‐risk dental procedures.MethodsThe antiviral activity of chlorine‐dioxide‐based oral rinse (OR) solutions was tested by pre‐incubating with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pseudovirus in a dosage‐dependent manner before transducing to human embryonic kidney epithelial (HEK293T‐ACE2) cells, which stably expresses ACE‐2 receptor. Viral entry was determined by measuring luciferase activity using a luminescence microplate reader. In the cell‐to‐cell fusion assay, effector Chinese hamster ovary (CHO‐K1) cells co‐expressing spike glycoprotein of SARS‐CoV‐2 and T7 RNA polymerase were pre‐incubated with the ORs before co‐culturing with the target CHO‐K1 cells co‐expressing human ACE2 receptor and luciferase gene. The luciferase signal was quantified 24 h after mixing the cells. Surface expression of SARS‐CoV‐2 spike glycoprotein and ACE‐2 receptor was confirmed using direct fluorescent imaging and quantitative cell‐ELISA. Finally, dosage‐dependent cytotoxic effects of ORs were evaluated at two different time points.ResultsA dosage‐dependent antiviral effect of the ORs was observed against SARS‐CoV‐2 cell entry and spike glycoprotein mediated cell‐to‐cell fusion. This demonstrates that ORs can be useful as a preprocedural step to reduce viral infectivity.ConclusionsChlorine‐dioxide‐based ORs have a potential benefit for reducing SARS‐CoV‐2 entry and spread.

The β2V287L nicotinic subunit linked to sleep-related epilepsy differently affects fast-spiking and regular spiking somatostatin-expressing neurons in murine prefrontal cortex.

Mutant subunits of the neuronal nicotinic ACh receptor (nAChR) can cause Autosomal Dominant Sleep-related Hypermotor Epilepsy (ADSHE), characterized by frontal seizures during non-rapid eye movement (NREM) sleep. We studied the cellular bases of the pathogenesis in brain slices from mice conditionally expressing the ADSHE-linked β2V287L nAChR subunit. β2V287L mice displayed minor structural alterations, except for a ~10% decrease of prefrontal cortex thickness. However, they showed a substantial decrease of the excitatory input to layer V fast-spiking (FS) interneurons, despite a concomitant increase in the number of glutamatergic terminals around the cell soma. Hence, prefrontal hyperexcitability may depend on a permanent impairment of surround inhibition. The effect disappeared when β2V287L was silenced until postnatal day 15th, suggesting that the transgene selectively affects the maturation of glutamatergic synapses on FS neurons. The other main population of interneurons in layer V was constituted by somatostatin-expressing regular spiking cells. When tested with 10µM nicotine, these displayed larger somatic nicotinic currents in transgenic mice. Thus, during wakefulness, activation of β2V287L-containing nAChRs by the high cholinergic tone may counteract hyperexcitability by promoting local inhibition by somatostatin-expressing cells and decreasing the effect of glutamatergic deficit in FS neurons. This interpretation was tested in networks disinhibited by 2μM bicuculline. Slices expressing β2V287L were more susceptible to develop synchronized activity in the absence of nicotine. Addition of the drug boosted excitability in the controls, but had little effect in β2V287L. Our findings suggest why NREM sleep favors ADSHE seizures and nicotine can be palliative in patients.

The Spike Protein of SARS-CoV-2 Impairs Lipid Metabolism and Increases Susceptibility to Lipotoxicity: Implication for a Role of Nrf2.

Coronavirus disease 2019 (COVID-19) patients show lipid metabolic alterations, but the mechanism remains unknown. In this study, we aimed to investigate whether the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impairs lipid metabolism in host cells. We generated a Spike cell line in HEK293 using the pcDNA vector carrying the Spike gene expression cassette. A control cell line was generated using the empty pcDNA vector. Gene expression profiles related to lipid metabolic, autophagic, and ferroptotic pathways were investigated. Palmitic acid (PA)-overload was used to assess lipotoxicity-induced necrosis. As compared with controls, the Spike cells showed a significant increase in lipid depositions in cell membranes as well as dysregulation of expression of a panel of molecules involving lipid metabolism, autophagy, and ferroptosis. The Spike cells showed an upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2), a multifunctional transcriptional factor, in response to PA. Furthermore, the Spike cells exhibited increased necrosis in response to PA-induced lipotoxicity compared to control cells in a time- and dose-dependent manner via ferroptosis, which could be attenuated by the Nrf2 inhibitor trigonelline. We conclude that the Spike protein impairs lipid metabolic and autophagic pathways in host cells, leading to increased susceptibility to lipotoxicity via ferroptosis which can be suppressed by a Nrf2 inhibitor. This data also suggests a central role of Nrf2 in Spike-induced lipid metabolic impairments.

ADAM10 and ADAM17 promote SARS‐CoV‐2 cell entry and spike protein‐mediated lung cell fusion

The severe‐acute‐respiratory‐syndrome‐coronavirus‐2 (SARS‐CoV‐2) is the causative agent of COVID‐19, but host cell factors contributing to COVID‐19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS‐CoV‐2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID‐19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS‐CoV‐2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease‐targeted inhibitors severely impair lung cell infection by the SARS‐CoV‐2 variants of concern alpha, beta, delta, and omicron and also reduce SARS‐CoV‐2 infection of primary human lung cells in a TMPRSS2 protease‐independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.

Discovery of Novel Activators of Large-Conductance Calcium-Activated Potassium Channels for the Treatment of Cerebellar Ataxia.

Impaired cerebellar Purkinje neuron firing resulting from reduced expression of large-conductance calcium-activated potassium (BK) channels is a consistent feature in models of inherited neurodegenerative spinocerebellar ataxia (SCA). Restoring BK channel expression improves motor function and delays cerebellar degeneration, indicating that BK channels are an attractive therapeutic target. Current BK channel activators lack specificity and potency and are therefore poor templates for future drug development. We implemented an automated patch clamp platform for high-throughput drug discovery of BK channel activators using the Nanion SyncroPatch 384PE system. We screened over 15,000 compounds for their ability to increase BK channel current amplitude under conditions of lower intracellular calcium that is present in disease. We identified several novel BK channel activators that were then retested on the SyncroPatch 384PE to generate concentration-response curves (CRCs). Compounds with favorable CRCs were subsequently tested for their ability to improve irregular cerebellar Purkinje neuron spiking, characteristic of BK channel dysfunction in SCA1 mice. We identified a novel BK channel activator, 4-chloro-N-(5-chloro-2-cyanophenyl)-3-(trifluoromethyl)benzene-1-sulfonamide (herein renamed BK-20), that exhibited a more potent half-maximal activation of BK current (pAC50 = 4.64) than NS-1619 (pAC50 = 3.7) at a free internal calcium concentration of 270 nM in a heterologous expression system and improved spiking regularity in SCA1 Purkinje neurons. BK-20 had no activity on small-conductance calcium-activated potassium (SK)1-3 channels but interestingly was a potent blocker of the T-type calcium channel, Cav3.1 (IC50 = 1.05 μM). Our work describes both a novel compound for further drug development in disorders with irregular Purkinje spiking and a unique platform for drug discovery in degenerative ataxias. SIGNIFICANCE STATEMENT: Motor impairment associated with altered Purkinje cell spiking due to dysregulation of large-conductance calcium-activated potassium (BK) channel expression and function is a shared feature of disease in many degenerative ataxias. BK channel activators represent an outstanding therapeutic agent for ataxia. We have developed a high-throughput platform to screen for BK channel activators and identified a novel compound that can serve as a template for future drug development for the treatment of these disabling disorders.

Immune Responses to SARS-CoV-2 Vaccination in Young Patients with Anti-CD19 Chimeric Antigen Receptor T Cell-Induced B Cell Aplasia

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are capable of inducing combined humoral and cellular immunity. Which effect is more relevant for their potent protective effects is unclear, but isolated T cell responses without seroconversion in healthy household members of individuals with Coronavirus disease 19 (COVID-19) suggest that T cell responses effectively protect against clinical infection. Oncologic patients have an outsize risk of unfavorable outcomes after SARS-CoV-2 infection and therefore were prioritized when vaccines first became available, although the quality of their immune response to vaccination was expected to be suboptimal, as has been confirmed in subsequent studies. Inherently, patients with anti-CD19 chimeric antigen receptor (CAR) T cell therapy-mediated B cell aplasia would be incapable of generating humoral responses, so that assessment of the vaccine-induced cellular immunity is all the more important to gauge whether the vaccine can induce meaningful protection. A salient difference between T cell and humoral responses is the former's relative impassiveness to mutations of the antigen, which is more relevant than ever since the advent of the omicron variant. The objective of this study was to assess the immune cell composition and spike protein-specific T cell responses before and after the first and second doses of SARS-CoV-2 mRNA vaccine in a cohort of juvenile CD19 CAR T cell therapy recipients with enduring B cell aplasia. The prospective study included all patients age >12 years diagnosed with multiply relapsed B cell precursor acute lymphoblastic leukemia and treated with anti-CD19 CAR T cell (CAR-T19) therapy in our center. The primary endpoint was the detection of cell-mediated and humoral responses to vaccine (flow cytometry and anti-S immunoglobulin G, respectively). Secondary endpoints included the incidence of vaccine-related grade 3 or 4 adverse events, exacerbation of graft-versus-host disease (GVHD), relapse, and the influence of the vaccine on CAR T cells and lymphocyte subsets. Even though one-half of the patients exhibited subnormal lymphocyte counts and marginal CD4/CD8 ratios, after 2 vaccinations all showed brisk T-cell responsiveness to spike protein, predominantly in the CD4 compartment, which quantitatively was well within the range of healthy controls. No severe vaccine-related grade 3 or 4 adverse events, GVHD exacerbation, or relapse was observed in our cohort. We posit that SARS-CoV-2 mRNA vaccines induce meaningful cellular immunity in patients with isolated B cell deficiency due to CAR-T19 therapy.

Interneuronal dynamics facilitate the initiation of spike block in cortical microcircuits.

Pyramidal cell spike block is a common occurrence in migraine with aura and epileptic seizures. In both cases, pyramidal cells experience hyperexcitation with rapidly increasing firing rates, major changes in electrochemistry, and ultimately spike block that temporarily terminates neuronal activity. In cortical spreading depression (CSD), spike block propagates as a slowly traveling wave of inactivity through cortical pyramidal cells, which is thought to precede migraine attacks with aura. In seizures, highly synchronized cortical activity can be interspersed with, or terminated by, spike block. While the identifying characteristic of CSD and seizures is the pyramidal cell hyperexcitation, it is currently unknown how the dynamics of the cortical microcircuits and inhibitory interneurons affect the initiation of hyperexcitation and subsequent spike block.We tested the contribution of cortical inhibitory interneurons to the initiation of spike block using a cortical microcircuit model that takes into account changes in ion concentrations that result from neuronal firing. Our results show that interneuronal inhibition provides a wider dynamic range to the circuit and generally improves stability against spike block. Despite these beneficial effects, strong interneuronal firing contributed to rapidly changing extracellular ion concentrations, which facilitated hyperexcitation and led to spike block first in the interneuron and then in the pyramidal cell. In all cases, a loss of interneuronal firing triggered pyramidal cell spike block. However, preventing interneuronal spike block was insufficient to rescue the pyramidal cell from spike block. Our data thus demonstrate that while the role of interneurons in cortical microcircuits is complex, they are critical to the initiation of pyramidal cell spike block. We discuss the implications that localized effects on cortical interneurons have beyond the isolated microcircuit and their contribution to CSD and epileptic seizures.

Aberrant Phase Precession of Lateral Septal Cells in a Maternal Immune Activation Model of Schizophrenia Risk May Disrupt the Integration of Location with Reward.

Spatial memory and reward processing are known to be disrupted in schizophrenia. Since the lateral septum (LS) may play an important role in the integration of location and reward, we examined the effect of maternal immune activation (MIA), a known schizophrenia risk factor, on spatial representation in the rat LS. In support of a previous study, we found that spatial location is represented as a phase code in the rostral LS of adult male rats, so that LS cell spiking shifts systematically against the phase of the hippocampal, theta-frequency, local field potential as an animal moves along a track toward a reward (phase precession). Whereas shallow precession slopes were observed in control group cells, they were steeper in the MIA animals, such that firing frequently precessed across several theta cycles as the animal moved along the length of the apparatus, with subsequent ambiguity in the phase representation of location. Furthermore, an analysis of the phase trajectories of the control group cells revealed that the population tended to converge toward a common firing phase as the animal approached the reward location. This suggested that phase coding in these cells might signal both reward location and the distance to reward. By comparison, the degree of phase convergence in the MIA-group cells was weak, and the region of peak convergence was distal to the reward location. These findings suggest that a schizophrenia risk factor disrupts the phase-based encoding of location-reward relationships in the LS, potentially smearing reward representations across space.SIGNIFICANCE STATEMENT It is unclear how spatial or contextual information generated by hippocampal cells is converted to a code that can be used to signal reward location in regions, such as the VTA. Here we provide evidence that the firing phase of cells in the lateral septum, a region that links the two areas, may code reward location in the firing phase of cells. This phase coding is disrupted in a maternal immune activation model of schizophrenia risk such that representations of reward may be smeared across space in maternal immune activation animals. This could potentially underlie erroneous reward processing and misattribution of salience in schizophrenia.

Synchronous spiking of cerebellar Purkinje cells during control of movements

SignificanceThe information that one region of the brain transmits to another is usually viewed through the lens of firing rates. However, if the output neurons could vary the timing of their spikes, then, through synchronization, they would spotlight information that may be critical for control of behavior. Here we report that, in the cerebellum, Purkinje cell populations that share a preference for error convey, to the nucleus, when to decelerate the movement, by reducing their firing rates and temporally synchronizing the remaining spikes.

Spike-timing-dependent plasticity rewards synchrony rather than causality.

Spike-timing-dependent plasticity (STDP) is a candidate mechanism for information storage in the brain, but the whole-cell recordings required for the experimental induction of STDP are typically limited to 1 h. This mismatch of time scales is a long-standing weakness in synaptic theories of memory. Here we use spectrally separated optogenetic stimulation to fire precisely timed action potentials (spikes) in CA3 and CA1 pyramidal cells. Twenty minutes after optogenetic induction of STDP (oSTDP), we observed timing-dependent depression (tLTD) and timing-dependent potentiation (tLTP), depending on the sequence of spiking. As oSTDP does not require electrodes, we could also assess the strength of these paired connections three days later. At this late time point, late tLTP was observed for both causal (CA3 before CA1) and anticausal (CA1 before CA3) timing, but not for asynchronous activity patterns (Δt = 50 ms). Blocking activity after induction of oSTDP prevented stable potentiation. Our results confirm that neurons wire together if they fire together, but suggest that synaptic depression after anticausal activation (tLTD) is a transient phenomenon.

High-frequency oscillations and sequence generation in two-population models of hippocampal region CA1.

Hippocampal sharp wave/ripple oscillations are a prominent pattern of collective activity, which consists of a strong overall increase of activity with superimposed (140 − 200 Hz) ripple oscillations. Despite its prominence and its experimentally demonstrated importance for memory consolidation, the mechanisms underlying its generation are to date not understood. Several models assume that recurrent networks of inhibitory cells alone can explain the generation and main characteristics of the ripple oscillations. Recent experiments, however, indicate that in addition to inhibitory basket cells, the pattern requires in vivo the activity of the local population of excitatory pyramidal cells. Here, we study a model for networks in the hippocampal region CA1 incorporating such a local excitatory population of pyramidal neurons. We start by investigating its ability to generate ripple oscillations using extensive simulations. Using biologically plausible parameters, we find that short pulses of external excitation triggering excitatory cell spiking are required for sharp/wave ripple generation with oscillation patterns similar to in vivo observations. Our model has plausible values for single neuron, synapse and connectivity parameters, random connectivity and no strong feedforward drive to the inhibitory population. Specifically, whereas temporally broad excitation can lead to high-frequency oscillations in the ripple range, sparse pyramidal cell activity is only obtained with pulse-like external CA3 excitation. Further simulations indicate that such short pulses could originate from dendritic spikes in the apical or basal dendrites of CA1 pyramidal cells, which are triggered by coincident spike arrivals from hippocampal region CA3. Finally we show that replay of sequences by pyramidal neurons and ripple oscillations can arise intrinsically in CA1 due to structured connectivity that gives rise to alternating excitatory pulse and inhibitory gap coding; the latter denotes phases of silence in specific basket cell groups, which induce selective disinhibition of groups of pyramidal neurons. This general mechanism for sequence generation leads to sparse pyramidal cell and dense basket cell spiking, does not rely on synfire chain-like feedforward excitation and may be relevant for other brain regions as well.

Visualizing a Field of Research for the Coronavirus Replication in Humans with Knowledge Mapping: Evidence from Web of Science.

BackgroundThe outbreak of COVID-19 sweeping the globe in 2020 has caused widespread fear and threatened global health security. Compared to SARS and MERS, COVID-19 also causes severe respiratory diseases and even fatal diseases but have many differences, such as the unidentified gene sequence and replication mechanism. From SARS to MERS, and then to COVID-19, coronaviruses have significant variations in host adaptation, virus evolution, infectivity, spread, and pathogenicity due to its unique replication mechanism.MethodsA field of research for the coronavirus replication in humans was visualized with a database covering 9177 kinds of literature in Web of Science from 2002 through October 2021 to provide cognitive direction for the epidemic situation of virus infection. Knowledge Mapping by CiteSpace and Bibliometrix Package in R Software was drawn to depict the underlying features of viral replication and changing trends of studies, with these analyses including co-citation, density visualization, keyword clustering, and time zone.ResultsThe keyword frequencies of "replication," ''infection," and ''spike protein" repeatedly appeared in published papers. Coronavirus can promote or inhibit apoptosis, depending on the balance between viral protein and apoptotic factors. When the living environment of cells is irreversibly damaged by the virus, cells have to start the apoptosis mechanism to prevent the replication, transmission, and spread of the virus. The replication, assembly and transmission of coronavirus can inhibit cells from entering the apoptosis prematurely with the fusion of spike protein and cell receptor in human.ConclusionOur results indicated that "viral infection," spike protein," and "mutation" might be future research hotspots on coronavirus replication in humans. The attention should be paid to the mutations of S protein and these mutants carrying mutations.Graphical abstract

Rapid odor processing by layer 2 subcircuits in lateral entorhinal cortex.

Olfactory information is encoded in lateral entorhinal cortex (LEC) by two classes of layer 2 (L2) principal neurons: fan and pyramidal cells. However, the functional properties of L2 cells and how they contribute to odor coding are unclear. Here, we show in awake mice that L2 cells respond to odors early during single sniffs and that LEC is essential for rapid discrimination of both odor identity and intensity. Population analyses of L2 ensembles reveal that rate coding distinguishes odor identity, but firing rates are only weakly concentration dependent and changes in spike timing can represent odor intensity. L2 principal cells differ in afferent olfactory input and connectivity with inhibitory circuits and the relative timing of pyramidal and fan cell spikes provides a temporal code for odor intensity. Downstream, intensity is encoded purely by spike timing in hippocampal CA1. Together, these results reveal the unique processing of odor information by LEC subcircuits and highlight the importance of temporal coding in higher olfactory areas.

Coumarins and Quinolones as Effective Multiple Targeted Agents Versus Covid-19: An In Silico Study.

The Covid-19 virus emerged a few months ago in China, and infections rapidly escalated into a pandemic. To date, there is no selective antiviral agent for the management of pathologies associated with covid-19 and the need for an effective agent against it is essential. In this work, two home-made databases from synthetic quinolines and coumarins were virtually docked against viral proteases (3CL and PL), human cell surface proteases (TMPRSS2 and furin) and spike proteins (S1 and S2). Chloroquine, a reference drug without a clear mechanism against coronavirus was also docked on mentioned targets and the binding affinities compared with title compounds. The best compounds of synthetic coumarins and quinolines for each target were determined. All compounds against all targets showed binding affinity between -5.80 to -8.99 kcal/mol in comparison with the FDA-approved drug, Chloroquine, with binding affinity of -5.7 to -7.98 kcal/mol. Two compounds, quinoline-1 and coumarin-24, were found to be effective on three targets - S2, TMPRSS2 and furin - simultaneously, with good predicted affinity between -7.54 to -8.85 kcal/mol. In silico ADME studies also confirmed good oral absorption for them. Furthermore, PASS prediction was calculated and coumarin-24 had higher probable activity (Pa) than probable inactivity (Pi) with acceptable protease inhibitory as well as good antiviral activity against Hepatitis C virus (HCV), Human immunodeficiency virus (HIV) and influenza. Quinoline-1 and Coumarin-24 have the potential to be used against Covid-19. Hence these agents could be useful in combating covid-19 infection after further in vitro and in vivo studies.

Secreted Trimeric Chikungunya Virus Spikes from Insect Cells: Production, Purification, and Glycosylation Status

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The CHIKV envelope glycoproteins E1 and E2 are important immunogens; therefore, the aim of this study is to produce trimeric CHIKV spikes in insect cells using the baculovirus expression system. The CHIKV E1 and E2 ectodomains were covalently coupled by a flexible linker that replaces the 6K transmembrane protein. The C-terminal E1 transmembrane was replaced by a Strep-tag II for the purification of secreted spikes from the culture fluid. After production in Sf9 suspension cells (product yields of 5.8–7.6 mg/L), the CHIKV spikes were purified by Strep-Tactin affinity chromatography, which successfully cleared the co-produced baculoviruses. Bis(sulfosuccinimidyl)suberate cross-linking demonstrated that the spikes are secreted as trimers. PNGase F treatment showed that the spikes are glycosylated. LC–MS/MS-based glycoproteomic analysis confirmed the glycosylation and revealed that the majority are of the mannose- or hybrid-type N-glycans and <2% have complex-type N-glycans. The LC –MS/MS analysis also revealed three O-glycosylation sites in E1. In conclusion, the trimeric, glycosylated CHIKV spikes have been successfully produced in insect cells and are now available for vaccination studies.

Electrical response of retinal ganglion cells in an N-methyl-N-nitrosourea-induced retinal degeneration porcine model

Retinal prosthesis is regarded as the treatment for vision restoration in the blind with retinal degeneration (RD) due to the loss of photoreceptors. A strategy for retinal prosthesis is to electrically activate surviving neurons. The retina’s response to electrical stimulation in a larger RD model has not been studied yet. Therefore, in this study, we investigated electrically evoked retinal responses in a previously validated N-methyl-N-nitrosourea (MNU)-induced porcine RD model. Electrically evoked responses were evaluated based on the number of retinal ganglion cell (RGC) spikes via multichannel recordings. Stimulation pulses were applied to degenerative and wild-type retinas with pulse modulation. Compared to wild-type retinas, degenerative retinas showed higher threshold values of pulse amplitude and pulse duration. The rate of increase in the number of RGC spikes relative to stimulus intensity was lower in degenerative retinas than in normal retinas. In severely degenerated retinas, few RGCs showed electrically evoked spikes. Our results suggest that the degenerative porcine retina requires a higher charge than the normal porcine retina. In the early stage of RD, it is easier to induce RGC spikes through electrical stimulation using retinal prosthesis; however, when the degeneration is severe, there may be difficulty recovering patient vision.

Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456–490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36–39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function.

FISH By Imaging Flow Cytometry in CLL for Diagnosis and MRD Assessment

▪Chronic lymphocytic leukaemia is a genetically heterogeneous disease with treatment and prognosis varying based on chromosomal abnormalities. These are detectable in up to 80% of cases when tested on the nuclei of interphase cells by fluorescence in situ hybridisation (FISH). Despite the clinical importance of FISH in management, as only up to 200 nuclei are generally assessed, it is not suitable for minimal residual disease (MRD) assessment. Since clinical decisions are based on detection thresholds of 10 -4, MRD assays are restricted to flow cytometry and molecular based assessment. Here we have explored the utility of a cutting-edge automated imaging flow cytometry method that incorporates cell immunophenotype and FISH (“immuno-flowFISH”) to detect chromosomal abnormalities in CLL.Aims: Our aim was to determine the capability of immuno-flowFISH using imaging flow cytometry to detect del(17p) and +12 in CLL, and, the lowest limit of detection. We hypothesized that this integrated automated immuno-flowFISH method would be suitable for MRD assessment of CLL.Methods: Blood from 75 patients with CLL, at diagnosis or on therapy, was analysed. For MRD studies, cells from the CI cell line were spiked into normal blood at concentrations of 0.001 - 10%. After red cell lysis, samples were incubated with CD3, CD5 and CD19 fluorophore-conjugated antibodies (fluorophores: BV480, BV605, AF647). Following fixation and membrane permeabilization, DNA was denatured at 78 oC for 5 mins. FISH probes to 17p12 and centromeres of chromosomes 12 and 17 were added and hybridized for 24 hours at 42 oC. Nuclei were then stained with SYTOX AADvanced and up to 600,000 cells acquired on the Amnis ® ImageStream ®XMk II imaging flow cytometer. Digital images (x60 objective) and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying cell nuclei. IDEAS was then used to assess the number and percent CD19/CD5-positive CLL cells with FISH abnormalities.Results: Between 10,000 and 600,000 cells (mean 60,000) were acquired. CLL (CD19/CD5-positive) and T- (CD3/CD5-positive) cells could be clearly identified by their immunophenotype and assessed individually for probe signals. FISH signals were identifiable on the digital images as specific “spots” overlying the SYTOX AADvanced nuclear stain. The IDEAS software could enumerate the number of FISH spots per cell and this was confirmed by quantitative mean channel fluorescence intensity for each probe. A chromosome 12 or 17 abnormality was detected in 23 of the 75 CLL cases. Of these, 10 cases had only one 17p signal (but 2 for the centromere of chromosome 17), indicative of del(17p). Del(17p) was detected in 2-35% of CD19/CD5-positive cells (i.e. 0.4-23% or 270-35,441 of all cells), the lowest seen in a patient on cytoreductive therapy. In 13/75 cases, there were 3 FISH signals for CEP12, consistent with trisomy 12 (+12) in 0.1-46% of all cells analysed; the lowest number of 0.1% was when 26 out of 26,000 cells analysed were CD19/CD5-positive and had +12. We also performed multi-FISH, incorporating CEP12, CEP17 and 17p probes together with the CD3, CD5 and CD19 antibodies. This required 7-fluorophores (antibodies, probes and nucleus) and confirmed the ability to detect del(17p) and chromosome 12 copy number simultaneously in a single analysis. Spiking of CI CLL cells into normal blood demonstrated that +12 could be detected to a lowest limit of 10 -5. In all analyses, CLL cells had normal diploid spots for the control CEP17 probe, and the CD3/CD5-positive T cells had dual signals for CEP12, CEP17 and 17p12 probes on numerical analysis and on digital imagery.Conclusion: This study of confirms that high-throughput automated imaging flow cytometry, integrating FISH and immunophenotyping, can detect chromosomal defects in CLL. The lowest limit of detection for a FISH-detectable abnormality was 10 -5. This high sensitivity and specificity exceeds current clinical practice (10 -4), and was achieved through the analysis of many thousands of cells and positive identification of CLL cells based on their phenotype. This immuno-flowFISH method does not require any prior cell separation and is automated. It adds a new dimension to chromosomal analysis in CLL, both at diagnosis and for MRD monitoring. The high precision and specificity of immuno-flowFISH illustrates that this has a real place as a new MRD assessment tool for CLL. DisclosuresNo relevant conflicts of interest to declare.

Bayesian Algorithmic Decoding of Acceleration and Speed Software (BADASS)

<h2>Abstract</h2> An important question in neuroscience is how the brain encodes the environment and the self, and how accurate these codes are. Bayesian decoding techniques have been used to determine the position of an animal based on neuron spike rate in the brain's hippocampus. In this software package, Bayesian Algorithmic Decoding of Acceleration and Speed Software (BADASS), we expand on existing decoding algorithms to decode speed and acceleration using cell spiking. BADASS can be used to determine the extent of speed and acceleration coding in different brain regions during movement, as well as during periods of memory consolidation and planning.