Electron tomography visualization of HIV-1 virions trapped by fusion inhibitors to host cells in infected tissues.

HIV-1 delivers its genetic material to infect a cell after fusion of the viral and host cell membranes, which takes place after the viral envelope (Env) binds host receptor and co-receptor proteins. Binding of host receptor CD4 to Env results in conformational changes that allow interaction with a host co-receptor (CCR5 or CXCR4). Further conformational rearrangements result in an elongated pre-hairpin intermediate structure in which Env is anchored to the viral membrane by its transmembrane region and to the host cell membrane by its fusion peptide. Although budding virions can be readily imaged by electron tomography (ET) of HIV-1-infected tissues and cultured cells, virions that are fusing (attached to host cells via pre-hairpin intermediates) are not normally visualized, perhaps because the process of membrane fusion is too fast to capture by EM. To image virions during fusion, we used fusion inhibitors to prevent downstream conformational changes in Env that lead to membrane fusion, thereby trapping HIV-1 virions linked to target cells by prehairpin intermediates. ET of HIV-1 pseudovirions bound to CD4+/CCR5+ TZM-bl cells revealed presumptive pre-hairpin intermediates as 2-4 narrow spokes linking a virion to the cell surface. To extend these results to a more physiological setting, we used ET to image tissues and organs derived from humanized bone marrow, liver, thymus (BLT) mice infected with HIV-1 and then treated with CPT31, a high-affinity D-peptide fusion inhibitor linked to cholesterol. Trapped HIV-1 virions were found in all tissues studied (small intestine, mesenteric lymph nodes, spleen, and bone marrow), and spokes representing pre-hairpin intermediates linking trapped virions to cell surfaces were similar in structure and number to those seen in the previous pseudovirus and cultured cell ET study.

Selective Activation of Peptide-Thioester Precursors for Templated Native Chemical Ligations.

Chemical protein synthesis enables access to proteins that would otherwise be difficult or impossible to obtain with traditional means such as recombinant expression. Chemoselective ligations provide the ability to join peptide segments prepared by solid-phase peptide synthesis. While native chemical ligation (NCL) is widely used, it is limited by the need for C-terminal thioesters with suitable reaction kinetics, properly placed native Cys or thiolated derivatives, and peptide segment solubility at low mM concentrations. Moreover, repetitive purifications to isolate ligated products are often yield-sapping, hampering efficiency and progress. In this work, we demonstrate the use of Controlled Activation of Peptides for Templated NCL (CAPTN). This traceless multi-segment templated NCL approach permits the one-pot synthesis of proteins by harnessing selective thioester activation and orthogonal conjugation chemistries to favor formation of full-length ligated product while minimizing side reactions. Importantly, CAPTN provides kinetic enhancements allowing ligations at sterically hindered junctions and low peptide concentrations. Additionally, this one-pot approach removes the need for intermediate purification. We report the synthesis of two E.coli ribosomal subunits S16 and S17 enabled by the chemical tools described herein. We anticipate that CAPTN will expedite the synthesis of valuable proteins and expand on templated approaches for chemical protein synthesis.

A synthetic delivery vector for mucosal vaccination.

The success of mRNA-based vaccines during the Covid-19 pandemic has highlighted the value of this new platform for vaccine development against infectious disease. However, the CD8+ T cell response remains modest with mRNA vaccines, and these do not induce mucosal immunity, which would be needed to prevent viral spread in the healthy population. To address this drawback, we developed a dendritic cell targeting mucosal vaccination vector, the homopentameric STxB. Here, we describe the highly efficient chemical synthesis of the protein, and its in vitro folding. This straightforward preparation led to a synthetic delivery tool whose biophysical and intracellular trafficking characteristics were largely indistinguishable from recombinant STxB. The chemical approach allowed for the generation of new variants with bioorthogonal handles. Selected variants were chemically coupled to several types of antigens derived from the mucosal viruses SARS-CoV-2 and type 16 human papillomavirus. Upon intranasal administration in mice, mucosal immunity, including resident memory CD8+ T cells and IgA antibodies was induced against these antigens. Our study thereby identifies a novel synthetic antigen delivery tool for mucosal vaccination with an unmatched potential to respond to an urgent medical need.

Clinical and Radiological Outcome of Osteoscopic-Assisted Treatment of Enchondroma in Hand with Artificial Bone Substitute or Bone Graft: A 7-Year Case Series and Literature Review.

Background: This study aims to look at the intermediate-term clinical, functional and radiological outcomes of patients with enchondroma in hand treated with osteoscopic-assisted curettage and artificial bone substitute or bone graft. The addition of osteoscopy allows direct visualisation of the bone cavity during and after curettage of tumour tissue without the need of creating a large opening in the bone cortex. This could lead to better clearance of tumour tissue and lower risk of iatrogenic fracture. Methods: A total of 11 patients who received surgery from December 2013 to November 2020 were retrospectively reviewed. All cases had histological diagnosis of enchondroma. Patients with a follow-up period of less than 3 months were excluded. The mean duration of follow-up was 20.9 months. For the clinical outcome, we measured the total active motion (TAM) and graded with Belsky score grip strength. For the functional outcome, the Quick Disabilities of the Arm, Shoulder and Hand Questionnaire (QuickDASH) score was used. For the radiological outcome, we evaluated the X-ray for bone cavity filling defect, new bone formation according to the system proposed by Tordai. Results: The mean TAM of patients was 257º. A total of 60% patients had Belsky score grading excellent, 40% patients had Belsky score grading good. The mean percentage of grip strength compared with the contralateral side was 86.2%. The mean QuickDASH score was 7.7. For the wound aesthetic rating by patients, 81.8% patients reported as excellent. For the radiological outcome, the postoperative X-ray of all patients showed bone filling defect less than 3 mm. The mean time to complete bone consolidation was 3.8 months. None of the patients showed any radiological signs of recurrence. Conclusions: Our study showed that patients with enchondromas in hand treated with this minimally invasive method demonstrated good functional and radiological outcome. Its application may also be extended into treating other benign bone lesions in hand. Level of Evidence: Level IV (Therapeutic).

Corrigendum: Neonatal NET-Inhibitory Factor improves survival in the cecal ligation and puncture model of polymicrobial sepsis by inhibiting neutrophil extracellular traps.

[This corrects the article DOI: 10.3389/fimmu.2022.1046574.].

A three-photon head-mounted microscope for imaging all layers of visual cortex in freely moving mice.

Advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2-g, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. The microscope had on-board photon detectors, robust to environmental light, and the arena lighting was timed to the end of each line-scan, enabling functional imaging of activity from cortical layer 4 and layer 6 neurons expressing jGCaMP7f in mice roaming a fully lit or dark arena. By comparing the neuronal activity measured from populations in these layers we show that activity in cortical layer 4 and layer 6 is differentially modulated by lit and dark conditions during free exploration.

Estimation of skeletal kinematics in freely moving rodents.

Forming a complete picture of the relationship between neural activity and skeletal kinematics requires quantification of skeletal joint biomechanics during free behavior; however, without detailed knowledge of the underlying skeletal motion, inferring limb kinematics using surface-tracking approaches is difficult, especially for animals where the relationship between the surface and underlying skeleton changes during motion. Here we developed a videography-based method enabling detailed three-dimensional kinematic quantification of an anatomically defined skeleton in untethered freely behaving rats and mice. This skeleton-based model was constrained using anatomical principles and joint motion limits and provided skeletal pose estimates for a range of body sizes, even when limbs were occluded. Model-inferred limb positions and joint kinematics during gait and gap-crossing behaviors were verified by direct measurement of either limb placement or limb kinematics using inertial measurement units. Together we show that complex decision-making behaviors can be accurately reconstructed at the level of skeletal kinematics using our anatomically constrained model.

Connectomic analysis of thalamus-driven disinhibition in cortical layer 4.

Sensory signals are transmitted via the thalamus primarily to layer 4 (L4) of the primary sensory cortices. While information about average neuronal connectivity in L4 is available, its detailed higher-order circuit structure is not known. Here, we used three-dimensional electron microscopy for a connectomic analysis of the thalamus-driven inhibitory network in L4. We find that thalamic input drives a subset of interneurons with high specificity, which in turn target excitatory neurons with subtype specificity. These interneurons create a directed disinhibitory network directly driven by the thalamic input. Neuronal activity recordings show that strong synchronous sensory activation yields about 1.5-fold stronger activation of star pyramidal cells than spiny stellates, in line with differential windows of opportunity for activation of excitatory neurons in the thalamus-driven disinhibitory circuit model. With this, we have identified a high degree of specialization of the microcircuitry in L4 of the primary sensory cortex.

Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies.

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a 'Serological Assay based on a Tri-part split-NanoLuc® (SATiN)' to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Here, we expand on our previous work and describe a reconfigured version of the SATiN assay, called Neutralization SATiN (Neu-SATiN), which measures neutralization activity of antibodies directly from convalescent or vaccinated sera. The results obtained with our assay and other neutralization assays are comparable but with significantly shorter preparation and run time for Neu-SATiN. As the assay is modular, we further demonstrate that Neu-SATiN enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

Improved Handling of Peptide Segments Using Side Chain-Based "Helping Hand" Solubilizing Tools.

Maintaining high, or even sufficient, solubility of every peptide segment in chemical protein synthesis (CPS) remains a critical challenge; insolubility of just a single peptide segment can thwart a total synthesis venture. Multiple approaches have been used to address this challenge, most commonly by employing a chemical tool to temporarily improve peptide solubility. In this chapter, we discuss chemical tools for introducing semipermanent solubilizing sequences (termed helping hands) at the side chains of Lys and Glu residues. We describe the synthesis, incorporation by Fmoc-SPPS, and cleavage conditions for utilizing these two tools. For Lys sites, we discuss the Fmoc-Ddap-OH dimedone-based linker, which is achiral, synthesized in one step, can be introduced directly at primary amines, and is removed using hydroxylamine (or hydrazine). For Glu sites, we detail the new Fmoc-SPPS building block, Fmoc-Glu(AlHx)-OH, which can be prepared in an efficient process over two purifications. Solubilizing sequences are introduced directly on-resin and later cleaved with palladium-catalyzed transfer under aqueous conditions to restore a native Glu side chain. These two chemical tools are straightforward to prepare and implement, and we anticipate continued usage in "difficult" peptide segments following the protocols described herein.

A joint NCBI and EMBL-EBI transcript set for clinical genomics and research.

Comprehensive genome annotation is essential to understand the impact of clinically relevant variants. However, the absence of a standard for clinical reporting and browser display complicates the process of consistent interpretation and reporting. To address these challenges, Ensembl/GENCODE1 and RefSeq2 launched a joint initiative, the Matched Annotation from NCBI and EMBL-EBI (MANE) collaboration, to converge on human gene and transcript annotation and to jointly define a high-value set of transcripts and corresponding proteins. Here, we describe the MANE transcript sets for use as universal standards for variant reporting and browser display. The MANE Select set identifies a representative transcript for each human protein-coding gene, whereas the MANE Plus Clinical set provides additional transcripts at loci where the Select transcripts alone are not sufficient to report all currently known clinical variants. Each MANE transcript represents an exact match between the exonic sequences of an Ensembl/GENCODE transcript and its counterpart in RefSeq such that the identifiers can be used synonymously. We have now released MANE Select transcripts for 97% of human protein-coding genes, including all American College of Medical Genetics and Genomics Secondary Findings list v3.0 (ref. 3) genes. MANE transcripts are accessible from major genome browsers and key resources. Widespread adoption of these transcript sets will increase the consistency of reporting, facilitate the exchange of data regardless of the annotation source and help to streamline clinical interpretation.

Neonatal NET-Inhibitory Factor improves survival in the cecal ligation and puncture model of polymicrobial sepsis by inhibiting neutrophil extracellular traps.

Introduction: Neutrophil extracellular traps (NETs) clear pathogens but may contribute Q8 pathogenically to host inflammatory tissue damage during sepsis. Innovative therapeutic agents targeting NET formation and their potentially harmful collateral effects remain understudied. Methods: We investigated a novel therapeutic agent, neonatal NET-Inhibitory Factor (nNIF), in a mouse model of experimental sepsis - cecal ligation and puncture (CLP). We administered 2 doses of nNIF (1 mg/ kg) or its scrambled peptide control intravenously 4 and 10 hours after CLP treatment and assessed survival, peritoneal fluid and plasma NET formation using the MPO-DNA ELISA, aerobic bacterial colony forming units (CFU) using serial dilution and culture, peritoneal fluid and stool microbiomes using 16S rRNA gene sequencing, and inflammatory cytokine levels using a multiplexed cytokine array. Meropenem (25 mg/kg) treatment served as a clinically relevant treatment for infection. Results: We observed increased 6-day survival rates in nNIF (73%) and meropenem (80%) treated mice compared to controls (0%). nNIF decreased NET formation compared to controls, while meropenem did not impact NET formation. nNIF treatment led to increased peritoneal fluid and plasma bacterial CFUs consistent with loss of NET-mediated extracellular microbial killing, while nNIF treatment alone did not alter the peritoneal fluid and stool microbiomes compared to vehicle-treated CLP mice. nNIF treatment also decreased peritoneal TNF-a inflammatory cytokine levels compared to scrambled peptide control. Furthermore, adjunctive nNIF increased survival in a model of sub-optimal meropenem treatment (90% v 40%) in CLP-treated mice. Discussion: Thus, our data demonstrate that nNIF inhibits NET formation in a translationally relevant mouse model of sepsis, improves survival when given as monotherapy or as an adjuvant with antibiotics, and may play an important protective role in sepsis.

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I).

The scope of proteins accessible to total chemical synthesis via native chemical ligation (NCL) is often limited by slow ligation kinetics. Here we describe Click-Assisted NCL (CAN), in which peptides are incorporated with traceless "helping hand" lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles. The resulting strain-promoted alkyne-azide cycloaddition (SPAAC) increases their effective concentration to greatly accelerate ligations. We demonstrate that copper(I) protects DBCO from acid-mediated rearrangement during acidic peptide cleavage, enabling direct production of DBCO synthetic peptides. Excitingly, triazole-linked model peptides ligated rapidly and accumulated little side product due to the fast reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in one pot. CAN is a useful method for overcoming challenging ligations involving sterically hindered junctions. Additionally, CAN is anticipated to be an important stepping stone toward a multisegment, one-pot, templated ligation system.

Visual pursuit behavior in mice maintains the pursued prey on the retinal region with least optic flow.

Mice have a large visual field that is constantly stabilized by vestibular ocular reflex (VOR) driven eye rotations that counter head-rotations. While maintaining their extensive visual coverage is advantageous for predator detection, mice also track and capture prey using vision. However, in the freely moving animal quantifying object location in the field of view is challenging. Here, we developed a method to digitally reconstruct and quantify the visual scene of freely moving mice performing a visually based prey capture task. By isolating the visual sense and combining a mouse eye optic model with the head and eye rotations, the detailed reconstruction of the digital environment and retinal features were projected onto the corneal surface for comparison, and updated throughout the behavior. By quantifying the spatial location of objects in the visual scene and their motion throughout the behavior, we show that the prey image consistently falls within a small area of the VOR-stabilized visual field. This functional focus coincides with the region of minimal optic flow within the visual field and consequently area of minimal motion-induced image-blur, as during pursuit mice ran directly toward the prey. The functional focus lies in the upper-temporal part of the retina and coincides with the reported high density-region of Alpha-ON sustained retinal ganglion cells.

Mice have a lot to keep an eye on. To survive, they need to dodge predators looming on land and from the skies, while also hunting down the small insects that are part of their diet. To do this, they are helped by their large panoramic field of vision, which stretches from behind and over their heads to below their snouts. To stabilize their gaze when they are on the prowl, mice reflexively move their eyes to counter the movement of their head: in fact, they are unable to move their eyes independently. This raises the question: what part of their large visual field of view do these rodents use when tracking a prey, and to what advantage? This is difficult to investigate, since it requires simultaneously measuring the eye and head movements of mice as they chase and capture insects. In response, Holmgren, Stahr et al. developed a new technique to record the precise eye positions, head rotations and prey location of mice hunting crickets in surroundings that were fully digitized at high resolution. Combining this information allowed the team to mathematically recreate what mice would see as they chased the insects, and to assess what part of their large visual field they were using. This revealed that, once a cricket had entered any part of the mice's large field of view, the rodents shifted their head ­ but not their eyes ­ to bring the prey into both eye views, and then ran directly at it. If the insect escaped, the mice repeated that behavior. During the pursuit, the cricket's position was mainly held in a small area of the mouse's view that corresponds to a specialized region in the eye which is thought to help track objects. This region also allowed the least motion-induced image blur when the animals were running forward. The approach developed by Holmgren, Stahr et al. gives a direct insight into what animals see when they hunt, and how this constantly changing view ties to what happens in the eyes. This method could be applied to other species, ushering in a new wave of tools to explore what freely moving animals see, and the relationship between behaviour and neural circuitry.

A glutamic acid-based traceless linker to address challenging chemical protein syntheses.

Native chemical ligation (NCL) enables the total chemical synthesis of proteins. However, poor peptide segment solubility remains a frequently encountered challenge. Here we introduce a traceless linker that can be temporarily attached to Glu side chains to overcome this problem. This strategy employs a new tool, Fmoc-Glu(AlHx)-OH, which can be directly installed using standard Fmoc-based solid-phase peptide synthesis. The incorporated residue, Glu(AlHx), is stable to a wide range of chemical protein synthesis conditions and is removed through palladium-catalyzed transfer under aqueous conditions. General handling characteristics, such as efficient incorporation, stability and rapid removal were demonstrated through a model peptide modified with Glu(AlHx) and a Lys6 solubilizing tag. Glu(AlHx) was incorporated into a highly insoluble peptide segment during the total synthesis of the bacteriocin AS-48. This challenging peptide was successfully synthesized and folded, and it has comparable antimicrobial activity to the native AS-48. We anticipate widespread use of this easy-to-use, robust linker for the preparation of challenging synthetic peptides and proteins.

Diarrheal pathogens trigger rapid evolution of the guanylate cyclase-C signaling axis in bats.

The pathogenesis of infectious diarrheal diseases is largely attributed to enterotoxins that cause dehydration by disrupting intestinal water absorption. We investigated patterns of genetic variation in mammalian guanylate cyclase-C (GC-C), an intestinal receptor targeted by bacterially encoded heat-stable enterotoxins (STa), to determine how host species adapt in response to diarrheal infections. Our phylogenetic and functional analysis of GC-C supports long-standing evolutionary conflict with diarrheal bacteria in primates and bats, with highly variable susceptibility to STa across species. In bats, we further show that GC-C diversification has sparked compensatory mutations in the endogenous uroguanylin ligand, suggesting an unusual scenario of pathogen-driven evolution of an entire signaling axis. Together, these findings suggest that conflicts with diarrheal pathogens have had far-reaching impacts on the evolution of mammalian gut physiology.

Enhancing native chemical ligation for challenging chemical protein syntheses.

Native chemical ligation has enabled the chemical synthesis of proteins for a wide variety of applications (e.g., mirror-image proteins). However, inefficiencies of this chemoselective ligation in the context of large or otherwise challenging protein targets can limit the practical scope of chemical protein synthesis. In this review, we focus on recent developments aimed at enhancing and expanding native chemical ligation for challenging protein syntheses. Chemical auxiliaries, use of selenium chemistry, and templating all enable ligations at otherwise suboptimal junctions. The continuing development of these tools is making the chemical synthesis of large proteins increasingly accessible.

Prevention and treatment of SHIVAD8 infection in rhesus macaques by a potent d-peptide HIV entry inhibitor.

Cholesterol-PIE12-trimer (CPT31) is a potent d-peptide HIV entry inhibitor that targets the highly conserved gp41 N-peptide pocket region. CPT31 exhibited strong inhibitory breadth against diverse panels of primary virus isolates. In a simian-HIV chimeric virus AD8 (SHIVAD8) macaque model, CPT31 prevented infection from a single high-dose rectal challenge. In chronically infected animals, CPT31 monotherapy rapidly reduced viral load by ∼2 logs before rebound occurred due to the emergence of drug resistance. In chronically infected animals with viremia initially controlled by combination antiretroviral therapy (cART), CPT31 monotherapy prevented viral rebound after discontinuation of cART. These data establish CPT31 as a promising candidate for HIV prevention and treatment.

Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate.

Fusion of HIV-1 with the membrane of its target cell, an obligate first step in virus infectivity, is mediated by binding of the viral envelope (Env) spike protein to its receptors, CD4 and CCR5/CXCR4, on the cell surface. The process of viral fusion appears to be fast compared with viral egress and has not been visualized by EM. To capture fusion events, the process must be curtailed by trapping Env-receptor binding at an intermediate stage. We have used fusion inhibitors to trap HIV-1 virions attached to target cells by Envs in an extended pre-hairpin intermediate state. Electron tomography revealed HIV-1 virions bound to TZM-bl cells by 2-4 narrow spokes, with slightly more spokes present when evaluated with mutant virions that lacked the Env cytoplasmic tail. These results represent the first direct visualization of the hypothesized pre-hairpin intermediate of HIV-1 Env and improve our understanding of Env-mediated HIV-1 fusion and infection of host cells.

Three-photon head-mounted microscope for imaging deep cortical layers in freely moving rats.

We designed a head-mounted three-photon microscope for imaging deep cortical layer neuronal activity in a freely moving rat. Delivery of high-energy excitation pulses at 1,320 nm required both a hollow-core fiber whose transmission properties did not change with fiber movement and dispersion compensation. These developments enabled imaging at >1.1 mm below the cortical surface and stable imaging of layer 5 neuronal activity for >1 h in freely moving rats performing a range of behaviors.