Molecular mechanism of choline and ethanolamine transport in humans.

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.

Molecular profiling of activated neurons by phosphorylated ribosome capture.

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue.

CCR3-dependent eosinophil recruitment is regulated by sialyltransferase ST3Gal-IV.

Eosinophil recruitment is a pathological hallmark of many allergic and helminthic diseases. Here, we investigated chemokine receptor CCR3-induced eosinophil recruitment in sialyltransferase St3gal4-/- mice. We found a marked decrease in eosinophil extravasation into CCL11-stimulated cremaster muscles and into the inflamed peritoneal cavity of St3gal4-/- mice. Ex vivo flow chamber assays uncovered reduced adhesion of St3gal4-/- compared to wild type eosinophils. Using flow cytometry, we show reduced binding of CCL11 to St3gal4-/- eosinophils. Further, we noted reduced binding of CCL11 to its chemokine receptor CCR3 isolated from St3gal4-/- eosinophils. This was accompanied by almost absent CCR3 internalization of CCL11-stimulated St3gal4-/- eosinophils. Applying an ovalbumin-induced allergic airway disease model, we found a dramatic reduction in eosinophil numbers in bronchoalveolar lavage fluid following intratracheal challenge with ovalbumin in St3gal4-deficient mice. Finally, we also investigated tissue-resident eosinophils under homeostatic conditions and found reduced resident eosinophil numbers in the thymus and adipose tissue in the absence of ST3Gal-IV. Taken together, our results demonstrate an important role of ST3Gal-IV in CCR3-induced eosinophil recruitment in vivo rendering this enzyme an attractive target in reducing unwanted eosinophil infiltration in various disorders including allergic diseases.

Flt3 agonist enhances immunogenicity of arenavirus vector-based simian immunodeficiency virus vaccine in macaques.

Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens are capable of inducing efficacious humoral and cellular immune responses in nonhuman primates. Several studies have evaluated the use of immune modulators to further enhance vaccine-induced T-cell responses. The hematopoietic growth factor Flt3L drives the expansion of various bone marrow progenitor populations, and administration of Flt3L was shown to promote expansion of dendritic cell populations in spleen and blood, which are targets of arenaviral vectors. Therefore, we evaluated the potential of Flt3 signaling to enhance the immunogenicity of arenaviral vaccines encoding SIV immunogens (SIVSME543 Gag, Env, and Pol) in rhesus macaques, with a rhesus-specific engineered Flt3L-Fc fusion protein. In healthy animals, administration of Flt3L-Fc led to a 10- to 100-fold increase in type 1 dendritic cells 7 days after dosing, with no antidrug antibody (ADA) generation after repeated dosing. We observed that administration of Flt3L-Fc fusion protein 7 days before arenaviral vaccine increased the frequency and activation of innate immune cells and enhanced T-cell activation with no treatment-related adverse events. Flt3L-Fc administration induced early innate immune activation, leading to a significant enhancement in magnitude, breadth, and polyfunctionality of vaccine-induced T-cell responses. The Flt3L-Fc enhancement in vaccine immunogenicity was comparable to a combination with αCTLA-4 and supports the use of safe and effective variants of Flt3L to augment therapeutic vaccine-induced T-cell responses.IMPORTANCEInduction of a robust human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell response through therapeutic vaccination is considered essential for HIV cure. Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens have demonstrated strong immunogenicity and efficacy in nonhuman primates. Here, we demonstrate that the immunogenicity of arenaviral vectors encoding SIV immunogens can be enhanced by administration of Flt3L-Fc fusion protein 7 days before vaccination. Flt3L-Fc-mediated increase in dendritic cells led to robust improvements in vaccine-induced T- and B-cell responses compared with vaccine alone, and Flt3L-Fc dosing was not associated with any treatment-related adverse events. Importantly, immune modulation by either Flt3L-Fc or αCTLA-4 led to comparable enhancement in vaccine response. These results indicate that the addition of Flt3L-Fc fusion protein before vaccine administration can significantly enhance vaccine immunogenicity. Thus, safe and effective Flt3L variants could be utilized as part of a combination therapy for HIV cure.

A replicating LCMV-based vaccine for the treatment of solid tumors.

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

Alternating Arenavirus Vector Immunization Generates Robust Polyfunctional Genotype Cross-Reactive Hepatitis B Virus-Specific CD8 T-Cell Responses and High Anti-Hepatitis B Surface Antigen Titers.

Hepatitis B Virus (HBV) is a major driver of infectious disease mortality. Curative therapies are needed and ideally should induce CD8 T cell-mediated clearance of infected hepatocytes plus anti-hepatitis B surface antigen (HBsAg) antibodies (anti-HBs) to neutralize residual virus. We developed a novel therapeutic vaccine using non-replicating arenavirus vectors. Antigens were screened for genotype conservation and magnitude and genotype reactivity of T cell response, then cloned into Pichinde virus (PICV) vectors (recombinant PICV, GS-2829) and lymphocytic choriomeningitis virus (LCMV) vectors (replication-incompetent, GS-6779). Alternating immunizations with GS-2829 and GS-6779 induced high-magnitude HBV T cell responses, and high anti-HBs titers. Dose schedule optimization in macaques achieved strong polyfunctional CD8 T cell responses against core, HBsAg, and polymerase and high titer anti-HBs. In AAV-HBV mice, GS-2829 and GS-6779 were efficacious in animals with low pre-treatment serum HBsAg. Based on these results, GS-2829 and GS-6779 could become a central component of cure regimens.

Localized chemogenetic silencing of inhibitory neurons: a novel mouse model of focal cortical epileptic activity.

Focal cortical epilepsies are frequently refractory to available anticonvulsant drug therapies. One key factor contributing to this state is the limited availability of animal models that allow to reliably study focal cortical seizures and how they recruit surrounding brain areas in vivo. In this study, we selectively expressed the inhibitory chemogenetic receptor, hM4D, in GABAergic neurons in focal cortical areas using viral gene transfer. GABAergic silencing using Clozapine-N-Oxide (CNO) demonstrated reliable induction of local epileptiform events in the electroencephalogram signal of awake freely moving mice. Anesthetized mice experiments showed consistent induction of focal epileptiform-events in both the barrel cortex (BC) and the medial prefrontal cortex (mPFC), accompanied by high-frequency oscillations, a known characteristic of human seizures. Epileptiform-events showed propagation indication with favored propagation pathways: from the BC on 1 hemisphere to its counterpart and from the BC to the mPFC, but not vice-versa. Lastly, sensory whisker-pad stimulation evoked BC epileptiform events post-CNO, highlighting the potential use of this model in studying sensory-evoked seizures. Combined, our results show that targeted chemogenetic inhibition of GABAergic neurons using hM4D can serve as a novel, versatile, and reliable model of focal cortical epileptic activity suitable for systematically studying cortical ictogenesis in different cortical areas.

Teletherapy Used to Breakdown Access Barriers.

The studies presented in this literature review reveal the numerous ways that teletherapy can be used to treat patients with mental health issues. The literature includes six research articles from published scientific journals that span from 2005 to 2020. The three types of telehealth therapy reviewed include mobile telehealth, telephone, and video technology. The six research articles focus on the ways that telehealth can reach communities of lower socioeconomic status (SES) and those suffering from access barriers. The benefits of teletherapy include cost savings, time efficiency, easier access, and a reduction in recidivism. Challenges include access barriers, financial difficulties, anxiety, and fear of stigmatization. Limitations of the studies presented include a lack of accessibility to internet and technology, privacy issues, and insurance coverage. Overall, results show that teletherapy provides an affordable, accessible alternative to traditional in-person mental health therapy, especially in reaching lower SES groups, Veterans, and patients with access restrictions.

Solid-State Oxidation of Alcohols in Gold-Coated Milling Vessels via Direct Mechanocatalysis.

This paper presents a novel approach for the selective oxidation of alcohols to their corresponding aldehydes through direct mechanocatalysis, employing a gold-coated milling vessel as catalyst and air as the oxidation agent. By adjusting milling frequency, media, and duration, high catalytic efficiencies and selectivities are achieved. Remarkably, yields of up to 99% are obtained for specific substrates, with a turnover number (TON) of 8200 and a turnover frequency (TOF) of 0.77 s-1, surpassing existing alternatives. Confirmation of the catalytic reaction indeed occurring on the milling tool surface was achieved through X-ray photoelectron spectroscopy (XPS).

Milling Medium-Free Suzuki Coupling by Direct Mechanocatalysis: From Mixer Mills to Resonant Acoustic Mixers.

Here we describe the development of a sustainable and cost-effective approach for catalytic cross-coupling reactions in mechanochemistry. It is found that the substrate's impact with the vessel wall alone is sufficient to initiate the reaction, thus indicating that milling balls function primarily as a mixing agent for direct mechanocatalytic Suzuki coupling. The absence of milling balls can be offset by adjusting the rheology using liquid-assisted grinding (LAG). The LAG sweet spot of 0.25 µL mg-1 is confirmed for both resonance acoustic mixers (RAMs) and ball-free mixer mills, and is higher than in the presence of milling balls. RAMs exhibit excellent performance in the Suzuki reaction, achieving yields of 90 % after 60 min and complete conversion after 90 min. The longevity of the milling vessel is significantly improved in a RAM, allowing for at least 20 reactions without deterioration.

SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function.

Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the (13)C(6)-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all organs tested. SILAC analysis from various organs lacking expression of beta1 integrin, beta-Parvin, or the integrin tail-binding protein Kindlin-3 confirmed their absence and disclosed a structural defect of the red blood cell membrane skeleton in Kindlin-3-deficient erythrocytes. The SILAC-mouse approach is a versatile tool by which to quantitatively compare proteomes from knockout mice and thereby determine protein functions under complex in vivo conditions.

Memory for social media images following traumatic brain injury.

PRIMARY OBJECTIVE: An emerging body of research examines the role of computer-mediated communication in supporting social connection in persons with traumatic brain injury (TBI). We examine the cognitive impacts of engaging with images posted to social media for persons with moderate-severe TBI. RESEARCH DESIGN: Prior work shows that after viewing social media posts, adults have better memory for posts when they generate a comment about the post. We examined if persons with TBI experience a memory benefit for commented-upon social media images similar to non-injured comparison participants. METHODS AND PROCEDURES: 53 persons with moderate-to-severe TBI and 52 non-injured comparison participants viewed arrays of real social media images and were prompted to comment on some of them. After a brief delay, a surprise two-alternative forced choice recognition memory test measured memory for these images. MAIN OUTCOMES AND RESULTS: Persons with TBI remembered social media images at above-chance levels and experienced a commenting-related memory boost much like non-injured comparison participants. CONCLUSIONS: These findings add to a growing literature on the potential benefits of social media use in individuals with TBI and point to the benefits of active engagement for memory in social media contexts in TBI.

PERCHING syndrome: Clinical presentation in the first African patient confirmed by clinical whole genome sequencing.

PERCHING syndrome is a rare multisystem developmental disorder caused by autosomal recessive (AR) variants (truncating and missense) in the Kelch-like family member 7 gene (KLHL7). We report the first phenotypic and molecular description of PERCHING syndrome in a patient from Central Africa. The patient presented multiple dysmorphic features in addition to neurological, respiratory, gastroenteric, and dysautonomic disorders. Clinical Whole Genome Sequencing in the proband and his mother identified two novel heterozygous variants in the KLHL7 gene, including a maternally inherited intronic variant (NM_001031710.2:c.793 + 5G > C) classified as Variant of Uncertain Significance and a frameshift stop gain variant (NM_001031710.2:c.944delG; p.Ser315ThrfsTer23) of unknown inheritance classified as likely pathogenic. Although the diagnosis was only evoked after genomic testing, the review of published patients suggests that this disease could be clinically recognizable and maybe considered as an encephalopathy. Our report will allow expanding the phenotypic and molecular spectrum of Perching syndrome.

The evolving landscape around genome editing in agriculture: Many countries have exempted or move to exempt forms of genome editing from GMO regulation of crop plants.

The EU and New Zealand are the only legislations where genome-edited plants are considered and regulated as GMOs while many other countries move to exempt genome-edited crops.

Bidirectional electromagnetic control of the hypothalamus regulates feeding and metabolism.

Targeted, temporally regulated neural modulation is invaluable in determining the physiological roles of specific neural populations or circuits. Here we describe a system for non-invasive, temporal activation or inhibition of neuronal activity in vivo and its use to study central nervous system control of glucose homeostasis and feeding in mice. We are able to induce neuronal activation remotely using radio waves or magnetic fields via Cre-dependent expression of a GFP-tagged ferritin fusion protein tethered to the cation-conducting transient receptor potential vanilloid 1 (TRPV1) by a camelid anti-GFP antibody (anti-GFP-TRPV1). Neuronal inhibition via the same stimuli is achieved by mutating the TRPV1 pore, rendering the channel chloride-permeable. These constructs were targeted to glucose-sensing neurons in the ventromedial hypothalamus in glucokinase-Cre mice, which express Cre in glucose-sensing neurons. Acute activation of glucose-sensing neurons in this region increases plasma glucose and glucagon, lowers insulin levels and stimulates feeding, while inhibition reduces blood glucose, raises insulin levels and suppresses feeding. These results suggest that pancreatic hormones function as an effector mechanism of central nervous system circuits controlling blood glucose and behaviour. The method we employ obviates the need for permanent implants and could potentially be applied to study other neural processes or used to regulate other, even dispersed, cell types.

Evaluating Provider Documentation of Suicide Risk Factors Among Youth Screening Positive for Suicide Risk in a Pediatric Emergency Department.

OBJECTIVE: Screening for suicidal ideation in the pediatric emergency department (ED) enhances recognition of suicidality among patients presenting with a nonmental health complaint. Little is known about the assessment of suicide risk factors and disposition among these patients. This study aimed to evaluate pediatric ED providers' documentation of suicide risk factors in this population. METHODS: We performed a retrospective cohort study of patients screening at risk for suicide on the Ask Suicide-Screening Questions tool. Demographic variables and risk factors for youth suicide were extracted from the electronic health record for eligible patients each month from January 1, 2019, to December 31, 2019. We compared risk factors using χ2 or Fisher exact test. RESULTS: In 2019, of the 7484 patients screened for suicide, 524 (7%) had a positive screen. Of 220 patient charts reviewed, no suicide risk factors were documented in 53.6% of encounters, and only 1 risk factor was documented in 18.2% of encounters. Substance use was the most frequently discussed risk factor, documented in 33.6% of encounters. History of nonsuicidal self-injury was documented in 11.8% of visits. Other risk factors were documented in fewer than 10% of at-risk patients. CONCLUSION: Pediatric ED providers do not routinely document risk factors for suicide in medical patients screening at risk. Although the Ask Suicide-Screening Questions is an important initial screen, a standardized secondary risk factor assessment is necessary for a more complete risk stratification for patients with suicidal ideation.

Supramodal neural networks support top-down processing of social signals.

The perception of facial and vocal stimuli is driven by sensory input and cognitive top-down influences. Important top-down influences are attentional focus and supramodal social memory representations. The present study investigated the neural networks underlying these top-down processes and their role in social stimulus classification. In a neuroimaging study with 45 healthy participants, we employed a social adaptation of the Implicit Association Test. Attentional focus was modified via the classification task, which compared two domains of social perception (emotion and gender), using the exactly same stimulus set. Supramodal memory representations were addressed via congruency of the target categories for the classification of auditory and visual social stimuli (voices and faces). Functional magnetic resonance imaging identified attention-specific and supramodal networks. Emotion classification networks included bilateral anterior insula, pre-supplementary motor area, and right inferior frontal gyrus. They were pure attention-driven and independent from stimulus modality or congruency of the target concepts. No neural contribution of supramodal memory representations could be revealed for emotion classification. In contrast, gender classification relied on supramodal memory representations in rostral anterior cingulate and ventromedial prefrontal cortices. In summary, different domains of social perception involve different top-down processes which take place in clearly distinguishable neural networks.

Complex effects of eslicarbazepine on inhibitory micro networks in chronic experimental epilepsy.

OBJECTIVE: Many antiseizure drugs (ASDs) act on voltage-dependent sodium channels, and the molecular basis of these effects is well established. In contrast, how ASDs act on the level of neuronal networks is much less understood. METHODS: In the present study, we determined the effects of eslicarbazepine (S-Lic) on different types of inhibitory neurons, as well as inhibitory motifs. Experiments were performed in hippocampal slices from both sham-control and chronically epileptic pilocarpine-treated rats. RESULTS: We found that S-Lic causes an unexpected reduction of feed-forward inhibition in the CA1 region at high concentrations (300 µM), but not at lower concentrations (100 µM). Concurrently, 300 but not 100 µM S-Lic significantly reduced maximal firing rates in putative feed-forward interneurons located in the CA1 stratum radiatum of sham-control and epileptic animals. In contrast, feedback inhibition was not inhibited by S-Lic. Instead, application of S-Lic, in contrast to previous data for other drugs like carbamazepine (CBZ), resulted in a lasting potentiation of feedback inhibitory post-synaptic currents (IPSCs) only in epileptic and not in sham-control animals, which persisted after washout of S-Lic. We hypothesized that this plasticity of inhibition might rely on anti-Hebbian potentiation of excitatory feedback inputs onto oriens-lacunosum moleculare (OLM) interneurons, which is dependent on Ca2+ -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Indeed, we show that blocking Ca2+ -permeable AMPA receptors completely prevents upmodulation of feedback inhibition. SIGNIFICANCE: These results suggest that S-Lic affects inhibitory circuits in the CA1 hippocampal region in unexpected ways. In addition, ASD actions may not be sufficiently explained by acute effects on their target channels, rather, it may be necessary to take plasticity of inhibitory circuits into account.

Altered Dynamics of Canonical Feedback Inhibition Predicts Increased Burst Transmission in Chronic Epilepsy.

Inhibitory interneurons, organized into canonical feedforward and feedback motifs, play a key role in controlling normal and pathological neuronal activity. We demonstrate prominent quantitative changes in the dynamics of feedback inhibition in a rat model of chronic epilepsy (male Wistar rats). Systematic interneuron recordings revealed a large decrease in intrinsic excitability of basket cells and oriens-lacunosum moleculare interneurons in epileptic animals. Additionally, the temporal dynamics of interneuron recruitment by recurrent feedback excitation were strongly altered, resulting in a profound loss of initial feedback inhibition during synchronous CA1 pyramidal activity. Biophysically constrained models of the complete feedback circuit motifs of normal and epileptic animals revealed that, as a consequence of altered feedback inhibition, burst activity arising in CA3 is more strongly converted to a CA1 output. This suggests that altered dynamics of feedback inhibition promote the transmission of epileptiform bursts to hippocampal projection areas.SIGNIFICANCE STATEMENT We quantitatively characterized changes of the CA1 feedback inhibitory circuit in a model of chronic temporal lobe epilepsy. This study shows, for the first time, that dynamic recruitment of inhibition in feedback circuits is altered and establishes the cellular mechanisms for this change. Computational modeling revealed that the observed changes are likely to systematically alter CA1 input-output properties leading to (1) increased seizure propagation through CA1 and (2) altered computation of synchronous CA3 input.

The Role of Donor Lymphocyte Infusion (DLI) in Post-Hematopoietic Cell Transplant (HCT) Relapse for Chronic Myeloid Leukemia (CML) in the Tyrosine Kinase Inhibitor (TKI) Era.

Treatment for relapse of chronic myeloid leukemia (CML) following hematopoietic cell transplantation (HCT) includes tyrosine kinase inhibitors (TKIs) with or without donor lymphocyte infusions (DLIs), but the most effective treatment strategy is unknown. This study was performed through the Center for International Blood and Marrow Transplant Research (CIBMTR) database. We retrospectively reviewed all patients reported to the CIBMTR registry from 2002 to 2014 who underwent HCT for CML and were alive 30 days postrelapse. A total of 215 HCT recipients relapsed and were analyzed in the following groups: (1) TKI alone (n = 128), (2) TKI with DLI (n = 48), and (3) DLI without TKI (n = 39). In multivariate analysis, disease status prior to HCT had a significant effect on overall survival (OS). Patients who received a DLI alone compared with a TKI with a DLI had inferior survival (hazard ratio, 2.28; 95% confidence interval, 1.23 to 4.24; P= .009). Those who received a TKI alone had similar survival compared with those who received a TKI with a DLI (P = .81). These data support that despite use of TKIs pretransplantation, TKI salvage therapy continues to provide significant survival following relapse in patients with CML following HCT. These data do not suggest that adding a DLI to a TKI adds an improvement in OS.