Psychedelic-inspired drug discovery using an engineered biosensor.

Ligands can induce G protein-coupled receptors (GPCRs) to adopt a myriad of conformations, many of which play critical roles in determining the activation of specific signaling cascades associated with distinct functional and behavioral consequences. For example, the 5-hydroxytryptamine 2A receptor (5-HT2AR) is the target of classic hallucinogens, atypical antipsychotics, and psychoplastogens. However, currently available methods are inadequate for directly assessing 5-HT2AR conformation both in vitro and in vivo. Here, we developed psychLight, a genetically encoded fluorescent sensor based on the 5-HT2AR structure. PsychLight detects behaviorally relevant serotonin release and correctly predicts the hallucinogenic behavioral effects of structurally similar 5-HT2AR ligands. We further used psychLight to identify a non-hallucinogenic psychedelic analog, which produced rapid-onset and long-lasting antidepressant-like effects after a single administration. The advent of psychLight will enable in vivo detection of serotonin dynamics, early identification of designer drugs of abuse, and the development of 5-HT2AR-dependent non-hallucinogenic therapeutics.

Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

Bispecific antibodies targeting two glycoproteins on SFTSV exhibit synergistic neutralization and protection in a mouse model.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high fatality rate of up to 30% caused by SFTS virus (SFTSV). However, no specific vaccine or antiviral therapy has been approved for clinical use. To develop an effective treatment, we isolated a panel of human monoclonal antibodies (mAbs). SF5 and SF83 are two neutralizing mAbs that recognize two viral glycoproteins (Gn and Gc), respectively. We found that their epitopes are closely located, and we then engineered them as several bispecific antibodies (bsAbs). Neutralization and animal experiments indicated that bsAbs display more potent protective effects than the parental mAbs, and the cryoelectron microscopy structure of a bsAb3 Fab-Gn-Gc complex elucidated the mechanism of protection. In vivo virus passage in the presence of antibodies indicated that two bsAbs resulted in less selective pressure and could efficiently bind to all single parental mAb-escape mutants. Furthermore, epitope analysis of the protective mAbs against SFTSV and RVFV indicated that they are all located on the Gn subdomain I, where may be the hot spots in the phleboviruses. Collectively, these data provide potential therapeutic agents and molecular basis for the rational design of vaccines against SFTSV infection.

NS2 induces an influenza A RNA polymerase hexamer and acts as a transcription to replication switch.

Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals.

An expanded palette of dopamine sensors for multiplex imaging in vivo.

Genetically encoded dopamine sensors based on green fluorescent protein (GFP) enable high-resolution imaging of dopamine dynamics in behaving animals. However, these GFP-based variants cannot be readily combined with commonly used optical sensors and actuators, due to spectral overlap. We therefore engineered red-shifted variants of dopamine sensors called RdLight1, based on mApple. RdLight1 can be combined with GFP-based sensors with minimal interference and shows high photostability, permitting prolonged continuous imaging. We demonstrate the utility of RdLight1 for receptor-specific pharmacological analysis in cell culture, simultaneous assessment of dopamine release and cell-type-specific neuronal activity and simultaneous subsecond monitoring of multiple neurotransmitters in freely behaving rats. Dual-color photometry revealed that dopamine release in the nucleus accumbens evoked by reward-predictive cues is accompanied by a rapid suppression of glutamate release. By enabling multiplexed imaging of dopamine with other circuit components in vivo, RdLight1 opens avenues for understanding many aspects of dopamine biology.

CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis.

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.

Ca2+/calmodulin binding to PSD-95 mediates homeostatic synaptic scaling down.

Postsynaptic density protein-95 (PSD-95) localizes AMPA-type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca2+ influx, Ca2+/calmodulin (Ca2+/CaM) binding to the N-terminus of PSD-95 mediates postsynaptic loss of PSD-95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD-95 N-terminus as important for binding to Ca2+/CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaMR126E, as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca2+/CaM to PSD-95 induced by a chronic increase in Ca2+ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission.

A Novel Doxorubicin Prodrug with GRP78 Recognition and Nucleus-Targeting Ability for Safe and Effective Cancer Therapy.

Glucose-regulated protein of 78 kDa (GRP78) has become an attractive and novel target for tumor therapy. Design and construction of powerful delivery systems that could efficiently transport doxorubicin (DOX) to a tumor-cell nucleus remains a formidable challenge for improving the tumor therapeutic index and mitigating side effects to normal tissues. Herein, a novel doxorubicin prodrug (NDP) with GRP78 recognition and nucleus-targeting ability was synthesized by a facile chemical route. NDP exhibited an enhanced antiproliferative activity against colorectal cancer cells and could efficiently enter the cell nucleus. Furthermore, it is inspiring to note that NDP displayed a much stronger inhibitory efficacy against the growth of colorectal cancer xenografts in nude mice than free DOX and showed superior in vivo safety. Together, the work provides a novel GRP78 and nucleus-targeting strategy, and the NDP holds great promise to be used as a potent and safe chemotherapeutic agent.

Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes.

Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15 Hz, 50 Hz, 75 Hz and 100 Hz) and at a flux density of 2 mT. Intracellular calcium concentration ([Ca(2+)]i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10 mM) was carried out to verify the function of the cardiac Na(+)/Ca(2+) exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca(2+)-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca(2+)]i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.

The binding and structural basis of fox ACE2 to RBDs from different sarbecoviruses.

Foxes are susceptible to SARS-CoV-2 in laboratory settings, and there have also been reports of natural infections of both SARS-CoV and SARS-CoV-2 in foxes. In this study, we assessed the binding capacities of fox ACE2 to important sarbecoviruses, including SARS-CoV, SARS-CoV-2, and animal-origin SARS-CoV-2 related viruses. Our findings demonstrated that fox ACE2 exhibits broad binding capabilities to receptor-binding domains (RBDs) of sarbecoviruses. We further determined the cryo-EM structures of fox ACE2 complexed with RBDs of SARS-CoV, SARS-CoV-2 prototype (PT), and Omicron BF.7. Through structural analysis, we identified that the K417 mutation can weaken the ability of SARS-CoV-2 sub-variants to bind to fox ACE2, thereby reducing the susceptibility of foxes to SARS-CoV-2 sub-variants. In addition, the Y498 residue in the SARS-CoV RBD plays a crucial role in forming a vital cation-π interaction with K353 in the fox ACE2 receptor. This interaction is the primary determinant for the higher affinity of the SARS-CoV RBD compared to that of the SARS-CoV-2 PT RBD. These results indicate that foxes serve as potential hosts for numerous sarbecoviruses, highlighting the critical importance of surveillance efforts.

Spike structures, receptor binding, and immune escape of recently circulating SARS-CoV-2 Omicron BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 sub-variants.

The recently emerged BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 variants have a growth advantage. In this study, we explore the structural bases of receptor binding and immune evasion for the Omicron BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 sub-variants. Our findings reveal that BA.2.86 exhibits strong receptor binding, whereas its JN.1 sub-lineage displays a decreased binding affinity to human ACE2 (hACE2). Through complex structure analyses, we observed that the reversion of R493Q in BA.2.86 receptor binding domain (RBD) plays a facilitating role in receptor binding, while the L455S substitution in JN.1 RBD restores optimal affinity. Furthermore, the structure of monoclonal antibody (mAb) S309 complexed with BA.2.86 RBD highlights the importance of the K356T mutation, which brings a new N-glycosylation motif, altering the binding pattern of mAbs belonging to RBD-5 represented by S309. These findings emphasize the importance of closely monitoring BA.2.86 and its sub-lineages to prevent another wave of SARS-CoV-2 infections.

Two noncompeting human neutralizing antibodies targeting MPXV B6 show protective effects against orthopoxvirus infections.

The recent outbreak of mpox epidemic, caused by monkeypox virus (MPXV), poses a new threat to global public health. Here, we initially assessed the preexisting antibody level to the MPXV B6 protein in vaccinia vaccinees born before the end of the immunization program and then identified two monoclonal antibodies (MAbs), hMB621 and hMB668, targeting distinct epitopes on B6, from one vaccinee. Binding assays demonstrate that both MAbs exhibit broad binding abilities to B6 and its orthologs in vaccinia (VACV), variola (VARV) and cowpox viruses (CPXV). Neutralizing assays reveal that the two MAbs showed potent neutralization against VACV. Animal experiments using a BALB/c female mouse model indicate that the two MAbs showed effective protection against VACV via intraperitoneal injection. Additionally, we determined the complex structure of B6 and hMB668, revealing the structural feature of B6 and the epitope of hMB668. Collectively, our study provides two promising antibody candidates for the treatment of orthopoxvirus infections, including mpox.

An economical and simple method for preparing highly permeable and chlorine-resistant reverse osmosis membranes with potential commercial applications.

The improvement in the overall efficiency of thin-film composite (TFC) reverse osmosis (RO) membranes is limited by their low permeability and sensitivity to degradation by chlorine. In the present study, polypiperazine (PIP), the commonly used amine monomer in preparing commercial TFC nanofiltration (NF) membranes, was used to regulate the m-phenylenediamine (MPD) based interfacial polymerization (IP) process. The results showed that addition of PIP optimized the micro-structure and surface properties of the polyamide (PA) layer. When the MPD and PIP mass ratio was 1 : 1, the TFCW-1:1 membrane exhibited 70% flux enhancement compared to pure MPD-based TFCW-1:0 membranes. Besides, the TFCW-1:1 membrane exhibited better chlorine-resistant performance since the NaCl rejection declined to just 3.8% while it was 11.3% for TFCW-1:0 membranes after immersion in 500 ppm NaClO solution for 48 h. Such improvement can be attributed to the increased number of unreacted amine groups and the thickness of the PA layer that PIP brought, which provided a sacrificial protective layer to consume the active chlorine, and thus maintain the integrity of the inner rejection layer. In all, the novelty and purpose of the present work is to find a more simple and scalable method to fabricate high-performance TFC RO membranes by using commonly, cheaply and frequently used materials.

Structural basis for receptor binding and broader interspecies receptor recognition of currently circulating Omicron sub-variants.

Multiple SARS-CoV-2 Omicron sub-variants, such as BA.2, BA.2.12.1, BA.4, and BA.5, emerge one after another. BA.5 has become the dominant strain worldwide. Additionally, BA.2.75 is significantly increasing in some countries. Exploring their receptor binding and interspecies transmission risk is urgently needed. Herein, we examine the binding capacities of human and other 28 animal ACE2 orthologs covering nine orders towards S proteins of these sub-variants. The binding affinities between hACE2 and these sub-variants remain in the range as that of previous variants of concerns (VOCs) or interests (VOIs). Notably, R493Q reverse mutation enhances the bindings towards ACE2s from humans and many animals closely related to human life, suggesting an increased risk of cross-species transmission. Structures of S/hACE2 or RBD/hACE2 complexes for these sub-variants and BA.2 S binding to ACE2 of mouse, rat or golden hamster are determined to reveal the molecular basis for receptor binding and broader interspecies recognition.

Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain.

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.

Intravenous functional gene transfer throughout the brain of non-human primates using AAV.

Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates (NHPs). Here we describe AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques with improved efficiency in the brain of multiple NHP species: marmoset, rhesus macaque, and green monkey. CAP-Mac is neuron-biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques, and is vasculature-biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver (1) functional GCaMP for ex vivo calcium imaging across multiple brain areas, and (2) a cocktail of fluorescent reporters for Brainbow-like labeling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. Given its capabilities for systemic gene transfer in NHPs, CAP-Mac promises to help unlock non-invasive access to the brain.

Secondhand Smoke Decreased Excitability and Altered Action Potential Characteristics of Cardiac Vagal Neurons in Mice.

Background: Secondhand smoke (SHS), a major indoor pollutant, is a significant risk factor for cardiovascular morbidity and mortality including arrhythmias and sudden cardiac death. Exposure to SHS can produce autonomic imbalance, as evidenced by reduced heart rate variability (HRV)-a clinical metric of cardiac vagal regulation. Currently, the mechanisms through which SHS changes the vagal preganglionic neuronal inputs to the heart to produce this remains unknown. Objectives: To characterize the effect of SHS on both the excitability and action potential (AP) characteristics of anatomically identified cardiac vagal neurons (CVNs) in the nucleus ambiguus and examine whether SHS alters small conductance calcium-activated potassium (SK) channel activity of these CVNs. Methods: Adult male mice were exposed to four weeks of filtered air or SHS (3 mg/m3) 6 h/day, 5 day/week. Using patch-clamp recordings on identified CVNs in brainstem slices, we determined neuronal excitability and AP characteristics with depolarizing step- and ramp-current injections. Results: Four weeks of SHS exposure reduced spiking responses to depolarizing current injections and increased AP voltage threshold in CVNs. Perfusion with apamin (20 nM) magnified these SHS-induced effects, suggesting reduced SK channel activity may serve to minimize the SHS-induced decreases in CVNs excitability. Medium afterhyperpolarization (a measurement of SK channel activity) was smaller in the SHS group, further supporting a lower SK channel activity. AP amplitude, rise rate, fast afterhyperpolarization amplitude (a measurement of voltage-gated channel activity), and decay rate were higher in the SHS group at membrane voltages more positive to 0 mV, suggesting altered inactivation properties of voltage-dependent channels underlying APs. Discussion: SHS exposure reduced neuronal excitability of CVNs with compensatory attenuation of SK channel activity and altered AP characteristics. Neuroplasticity of CVNs could blunt regulatory cardiac vagal signaling and contribute to the cardiovascular consequences associated with SHS exposure, including reduced HRV.

Cryo-EM structure of the varicella-zoster virus A-capsid.

Varicella-zoster virus (VZV), a member of the Alphaherpesvirinae subfamily, causes severe diseases in humans of all ages. The viral capsids play critical roles in herpesvirus infection, making them potential antiviral targets. Here, we present the 3.7-Å-resolution structure of the VZV A-capsid and define the molecular determinants underpinning the assembly of this complicated viral machinery. Overall, the VZV capsid has a similar architecture to that of other known herpesviruses. The major capsid protein (MCP) assembles into pentons and hexons, forming extensive intra- and inter-capsomer interaction networks that are further secured by the small capsid protein (SCP) and the heterotriplex. The structure reveals a pocket beneath the floor of MCP that could potentially be targeted by antiviral inhibitors. In addition, we identified two alphaherpesvirus-specific structural features in SCP and Tri1 proteins. These observations highlight the divergence of different herpesviruses and provide an important basis for developing antiviral drugs.

Cryo-EM Structure of the African Swine Fever Virus.

African swine fever virus (ASFV) is a large double-stranded DNA virus with an icosahedral multilayered structure. ASFV causes a lethal swine hemorrhagic disease and is currently responsible for widespread damage to the pork industry in Asia. Neither vaccines nor antivirals are available and the molecular characterization of the ASFV particle is outstanding. Here, we describe the cryogenic electron microscopy (cryo-EM) structure of the icosahedral capsid of ASFV at 4.6-Å. The ASFV particle consists of 8,280 copies of the major capsid protein p72, 60 copies of the penton protein, and at least 8,340 minor capsid proteins, of which there might be 3 different types. Like other nucleocytoplasmic large DNA viruses, the minor capsid proteins form a hexagonal network below the outer capsid shell, functioning as stabilizers by "gluing" neighboring capsomers together. Our findings provide a comprehensive molecular model of the ASFV capsid architecture that will contribute to the future development of countermeasures, including vaccines.

Robust Anticancer Efficacy of a Biologically Synthesized Tumor Acidity-Responsive and Autophagy-Inducing Functional Beclin 1.

As a potent autophagy inducer, Beclin 1 is essential for the initiation of autophagic cell death, and triggering extensive autophagy by targeted delivery of Beclin 1 to tumors has enormous potential to inhibit tumor growth. Yet, the therapeutic application of Beclin 1 is hampered by its inability to internalize into cells and nonselective biodistribution in vivo. To tackle this challenge, we employed a novel Beclin 1 delivery manner by constructing a functional protein (Trx-pHLIP-Beclin 1, TpB) composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP), and an evolutionarily conserved motif of Beclin 1. This protein could effectively transport Beclin 1 to breast and ovarian cancer cell lines under weakly acidic conditions (pH 6.5), markedly inhibit tumor cell growth and proliferation, and induce obvious autophagy. Furthermore, the in vivo antitumor efficacy of the functional Beclin 1 against an SKOV3 xenograft tumor mouse model was tested via intravenous injection. TpB preferentially accumulated in tumors and exhibited a significantly higher tumor growth inhibition than the nontargeted Beclin 1 control, whereas no overt side effects were observed. Taken together, this study sheds light on the potential application of TpB as a highly efficient yet safe antitumor agent for cancer treatment.