Optimized intravital three-photon imaging of intact mouse tibia links plasma cell motility to functional states.

Intravital deep bone marrow imaging is crucial to studying cellular dynamics and functions but remains challenging, and minimally invasive methods are needed. We employed a high pulse-energy 1650 nm laser to perform three-photon microscopy , reaching ≈400 μm depth in intact mouse tibia. Repetition rates of 3 and 4 MHz allowed us to analyze motility patterns of fast and rare cells within unperturbed marrow and to identify a bi-modal migratory behavior for plasma cells.

Mimicking opioid analgesia in cortical pain circuits.

The anterior cingulate cortex plays a pivotal role in the cognitive and affective aspects of pain perception. Both endogenous and exogenous opioid signaling within the cingulate mitigate cortical nociception, reducing pain unpleasantness. However, the specific functional and molecular identities of cells mediating opioid analgesia in the cingulate remain elusive.

Into the Wild: A novel wild-derived inbred strain resource expands the genomic and phenotypic diversity of laboratory mouse models.

The laboratory mouse has served as the premier animal model system for both basic and preclinical investigations for over a century. However, laboratory mice capture only a subset of the genetic variation found in wild mouse populations, ultimately limiting the potential of classical inbred strains to uncover phenotype-associated variants and pathways. Wild mouse populations are reservoirs of genetic diversity that could facilitate the discovery of new functional and disease-associated alleles, but the scarcity of commercially available, well-characterized wild mouse strains limits their broader adoption in biomedical research.

A nociceptive amygdala-striatal pathway for chronic pain aversion.

The basolateral amygdala (BLA) is essential for assigning positive or negative valence to sensory stimuli. Noxious stimuli that cause pain are encoded by an ensemble of ceptive BLA projection neurons (BLA ensemble). However, the role of the BLA ensemble in mediating behavior changes and the molecular signatures and downstream targets distinguishing this ensemble remain poorly understood.

generated antibodies guide thermostable ADDomer nanoparticle design for nasal vaccination and passive immunization against SARS-CoV-2.

Background: Due to COVID-19, pandemic preparedness emerges as a key imperative, necessitating new approaches to accelerate development of reagents against infectious pathogens.

Methods: Here, we developed an integrated approach combining synthetic, computational and structural methods with antibody selection and immunization to design, produce and validate nature-inspired nanoparticle-based reagents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Results: Our approach resulted in two innovations: (i) a thermostable nasal vaccine called ADDoCoV, displaying multiple copies of a SARS-CoV-2 receptor binding motif derived epitope and (ii) a multivalent nanoparticle superbinder, called Gigabody, against SARS-CoV-2 including immune-evasive variants of concern (VOCs).

Into the Wild: A novel wild-derived inbred strain resource expands the genomic and phenotypic diversity of laboratory mouse models.

The laboratory mouse has served as the premier animal model system for both basic and preclinical investigations for a century. However, laboratory mice capture a narrow subset of the genetic variation found in wild mouse populations. This consideration inherently restricts the scope of potential discovery in laboratory models and narrows the pool of potentially identified phenotype-associated variants and pathways.

Human OPRM1 and murine Oprm1 promoter driven viral constructs for genetic access to μ-opioidergic cell types.

With concurrent global epidemics of chronic pain and opioid use disorders, there is a critical need to identify, target and manipulate specific cell populations expressing the mu-opioid receptor (MOR). However, available tools and transgenic models for gaining long-term genetic access to MOR+ neural cell types and circuits involved in modulating pain, analgesia and addiction across species are limited. To address this, we developed a catalog of MOR promoter (MORp) based constructs packaged into adeno-associated viral vectors that drive transgene expression in MOR+ cells.

High-affinity CD8 variants enhance the sensitivity of pMHCI antigen recognition via low-affinity TCRs.

CD8 T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation.

Prolonged T-cell activation and long COVID symptoms independently associate with severe COVID-19 at 3 months.

Coronavirus disease-19 (COVID-19) causes immune perturbations which may persist long term, and patients frequently report ongoing symptoms for months after recovery. We assessed immune activation at 3-12 months post hospital admission in 187 samples from 63 patients with mild, moderate, or severe disease and investigated whether it associates with long COVID. At 3 months, patients with severe disease displayed persistent activation of CD4 and CD8 T-cells, based on expression of HLA-DR, CD38, Ki67, and granzyme B, and elevated plasma levels of interleukin-4 (IL-4), IL-7, IL-17, and tumor necrosis factor-alpha (TNF-α) compared to mild and/or moderate patients.

Hyperactive immature state and differential CXCR2 expression of neutrophils in severe COVID-19.

Neutrophils are vital in defence against pathogens, but excessive neutrophil activity can lead to tissue damage and promote acute respiratory distress syndrome. COVID-19 is associated with systemic expansion of immature neutrophils, but the functional consequences of this shift to immaturity are not understood. We used flow cytometry to investigate activity and phenotypic diversity of circulating neutrophils in acute and convalescent COVID-19 patients.

Ready and waiting to go.

Some T cells that have been activated by a herpesvirus can also respond to SARS-CoV-2, even if the original herpesvirus infection happened before the COVID-19 pandemic.

A novel Oprm1-Cre mouse maintains endogenous expression, function and enables detailed molecular characterization of μ-opioid receptor cells.

Key targets of both the therapeutic and abused properties of opioids are μ-opioid receptors (MORs). Despite years of research investigating the biochemistry and signal transduction pathways associated with MOR activation, we do not fully understand the cellular mechanisms underlying opioid addiction. Given that addictive opioids such as morphine, oxycodone, heroin, and fentanyl all activate MORs, and current therapies such as naloxone and buprenorphine block this activation, the availability of tools to mechanistically investigate opioid-mediated cellular and behavioral phenotypes are necessary.

An updated protocol for in vitro bovine embryo production.

Cattle embryos represent a useful model for understanding parts of human embryogenesis due to various biological similarities. We describe a protocol to mature and fertilize bovine oocytes followed by culture of resulting presumptive zygotes up until the blastocyst stage. Our protocol features a unique procedure for washing and moving oocytes and zygotes between their respective dishes using a cell strainer.

Rapid Evolution of the Fine-scale Recombination Landscape in Wild House Mouse (Mus musculus) Populations.

Meiotic recombination is an important evolutionary force and an essential meiotic process. In many species, recombination events concentrate into hotspots defined by the site-specific binding of PRMD9. Rapid evolution of Prdm9's zinc finger DNA-binding array leads to remarkably abrupt shifts in the genomic distribution of hotspots between species, but the question of how Prdm9 allelic variation shapes the landscape of recombination between populations remains less well understood.

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2.

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269).

Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells.

Background: Cytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence.

Methods: Here, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes.

Bioactive supplements influencing bovine in vitro embryo development.

Ovum pickup and in vitro production (IVP) of bovine embryos are replacing traditional multiple ovulation embryo transfer (MOET) as the primary means for generating transferable embryos from genetically elite sires and dams. However, inefficiencies in the IVP process limit the opportunities to produce large numbers of transferable embryos. Also, the post-transfer competency of IVP embryos is inferior to embryos produced by artificial insemination or MOET.

Cytokines That Serve as Embryokines in Cattle.

The term "embryokine" has been used to denote molecules produced by the endometrium, oviduct, or by embryo itself that will influence embryo development. Several cytokines have been identified as embryokines in cattle and other mammals. This review will describe how these cytokines function as embryokines, with special emphasis being placed on their actions on in vitro produced (IVP) bovine embryos.

Synthetic Peptides with Inadvertent Chemical Modifications Can Activate Potentially Autoreactive T Cells.

The human CD8 T cell clone 6C5 has previously been shown to recognize the -butyl-modified Bax peptide LLSY(3-Bu)FGTPT presented by HLA-A*02:01. This nonnatural epitope was likely created as a by-product of fluorenylmethoxycarbonyl protecting group peptide synthesis and bound poorly to HLA-A*02:01. In this study, we used a systematic approach to identify and characterize natural ligands for the 6C5 TCR.

CD8 coreceptor-mediated focusing can reorder the agonist hierarchy of peptide ligands recognized via the T cell receptor.

CD8 T cells are inherently cross-reactive and recognize numerous peptide antigens in the context of a given major histocompatibility complex class I (MHCI) molecule via the clonotypically expressed T cell receptor (TCR). The lineally expressed coreceptor CD8 interacts coordinately with MHCI at a distinct and largely invariant site to slow the TCR/peptide-MHCI (pMHCI) dissociation rate and enhance antigen sensitivity. However, this biological effect is not necessarily uniform, and theoretical models suggest that antigen sensitivity can be modulated in a differential manner by CD8.

Young infants exhibit robust functional antibody responses and restrained IFN-γ production to SARS-CoV-2.

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in 4 infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19.

Interleukin-6 supplementation improves post-transfer embryonic and fetal development of in vitro-produced bovine embryos.

The use of in vitro produced embryos in dairy and beef cattle has increased in recent years, but compromised post-transfer pregnancy success prevents producers from capturing all the benefits this technology can provide. This study explored whether supplementing interleukin-6 (IL6) during in vitro embryo development influences post-transfer development of the embryo-proper, fetus and placenta during early gestation in cattle. Slaughterhouse-derived cumulus oocyte complexes underwent IVM (day -1) and IVF (day 0).

Physical parameters of bovine activated oocytes and zygotes as predictors of development success.

The worldwide production of in vitro-produced embryos in livestock species continues to grow. The current gold standard for selecting quality oocytes and embryos is morphologic assessment, yet this method is subjective and varies based on experience. There is a need for a non-invasive, objective method of selecting viable oocytes and embryos.

Interleukin-6 promotes primitive endoderm development in bovine blastocysts.

Background: Interleukin-6 (IL6) was recently identified as an embryotrophic factor in bovine embryos, where it acts primarily to mediate inner cell mass (ICM) size. This work explored whether IL6 affects epiblast (EPI) and primitive endoderm (PE) development, the two embryonic lineages generated from the ICM after its formation. Nuclear markers for EPI (NANOG) and PE (GATA6) were used to differentiate the two cell types.

Post-acute COVID-19 associated with evidence of bystander T-cell activation and a recurring antibiotic-resistant bacterial pneumonia.

Here, we describe the case of a COVID-19 patient who developed recurring ventilator-associated pneumonia caused by that acquired increasing levels of antimicrobial resistance (AMR) in response to treatment. Metagenomic analysis revealed the AMR genotype, while immunological analysis revealed massive and escalating levels of T-cell activation. These were both SARS-CoV-2 and specific, and bystander activated, which may have contributed to this patient's persistent symptoms and radiological changes.