Metabolic programs of T cell tissue residency empower tumour immunity.

Tissue resident memory CD8+ T (TRM) cells offer rapid and long-term protection at sites of reinfection1. Tumour-infiltrating lymphocytes with characteristics of TRM cells maintain enhanced effector functions, predict responses to immunotherapy and accompany better prognoses2,3. Thus, an improved understanding of the metabolic strategies that enable tissue residency by T cells could inform new approaches to empower immune responses in tissues and solid tumours. Here, to systematically define the basis for the metabolic reprogramming supporting TRM cell differentiation, survival and function, we leveraged in vivo functional genomics, untargeted metabolomics and transcriptomics of virus-specific memory CD8+ T cell populations. We found that memory CD8+ T cells deployed a range of adaptations to tissue residency, including reliance on non-steroidal products of the mevalonate-cholesterol pathway, such as coenzyme Q, driven by increased activity of the transcription factor SREBP2. This metabolic adaptation was most pronounced in the small intestine, where TRM cells interface with dietary cholesterol and maintain a heightened state of activation4, and was shared by functional tumour-infiltrating lymphocytes in diverse tumour types in mice and humans. Enforcing synthesis of coenzyme Q through deletion of Fdft1 or overexpression of PDSS2 promoted mitochondrial respiration, memory T cell formation following viral infection and enhanced antitumour immunity. In sum, through a systematic exploration of TRM cell metabolism, we reveal how these programs can be leveraged to fuel memory CD8+ T cell formation in the context of acute infections and enhance antitumour immunity.

Neuroprotection in Cerebral Cortex Induced by the Pregnancy Hormone Estriol.

In multiple sclerosis (MS), demyelination occurs in the cerebral cortex, and cerebral cortex atrophy correlates with clinical disabilities. Treatments are needed in MS to induce remyelination. Pregnancy is protective in MS. Estriol is made by the fetoplacental unit, and maternal serum estriol levels temporally align with fetal myelination. Here, we determined the effect of estriol treatment on the cerebral cortex in the preclinical model of MS, experimental autoimmune encephalomyelitis (EAE). Estriol treatment initiated after disease onset decreased cerebral cortex atrophy. Neuropathology of the cerebral cortex showed increased cholesterol synthesis proteins in oligodendrocytes, more newly formed remyelinating oligodendrocytes, and increased myelin in estriol-treated EAE mice. Estriol treatment also decreased the loss of cortical layer V pyramidal neurons and their apical dendrites and preserved synapses. Together, estriol treatment after EAE onset reduced atrophy and was neuroprotective in the cerebral cortex.

Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease.

Molecular integrators, in contrast to real-time indicators, convert transient cellular events into stable signals that can be exploited for imaging, selection, molecular characterization, or cellular manipulation. Many integrators, however, are designed as complex multicomponent circuits that have limited robustness, especially at high, low, or nonstoichiometric protein expression levels. Here, we report a simplified design of the calcium and light dual integrator FLARE. Single-chain FLARE (scFLARE) is a single polypeptide chain that incorporates a transcription factor, a LOV domain-caged protease cleavage site, and a calcium-activated TEV protease that we designed through structure-guided mutagenesis and screening. We show that scFLARE has greater dynamic range and robustness than first-generation FLARE and can be used in culture as well as in vivo to record patterns of neuronal activation with 10-min temporal resolution.

Oral desensitization therapy for peanut allergy induces dynamic changes in peanut-specific immune responses.

BACKGROUND: The PALISADE study, an international, phase 3 trial of peanut oral immunotherapy (POIT) with AR101, resulted in desensitization in children and adolescents who were highly allergic to peanut. An improved understanding of the immune mechanism induced in response to food allergen immunotherapy would enable more informed and effective therapeutic strategies. Our main purpose was to examine the immunological changes in blood samples from a subset of peanut-allergic individuals undergoing oral desensitization immunotherapy with AR101. METHODS: Blood samples obtained as part of enrollment screening and at multiple time points during PALISADE study were used to assess basophil and CD4+ T-cell reactivity to peanut. RESULTS: The absence of clinical reactivity to the entry double-blinded placebo-controlled peanut challenge (DBPCFC) was accompanied by a significantly lower basophil sensitivity and T-cell reactivity to peanut compared with DBPCFC reactors. At baseline, peanut-reactive TH2A cells were observed in many but not all peanut-allergic patients and their level in peripheral blood correlates with T-cell reactivity to peanut and with serum peanut-specific IgE and IgG4 levels. POIT reshaped circulating peanut-reactive T-cell responses in a subset-dependent manner. Changes in basophil and T-cell responses to peanut closely paralleled clinical benefits to AR101 therapy and resemble responses in those with lower clinical sensitivity to peanut. However, no difference in peanut-reactive Treg cell frequency was observed between groups. CONCLUSION: Oral desensitization therapy with AR101 leads to decreased basophil sensitivity to peanut and reshapes peanut-reactive T effector cell responses supporting its potential as an immunomodulatory therapy.

Systems scale interactive exploration reveals quantitative and qualitative differences in response to influenza and pneumococcal vaccines.

Systems immunology approaches were employed to investigate innate and adaptive immune responses to influenza and pneumococcal vaccines. These two non-live vaccines show different magnitudes of transcriptional responses at different time points after vaccination. Software solutions were developed to explore correlates of vaccine efficacy measured as antibody titers at day 28. These enabled a further dissection of transcriptional responses. Thus, the innate response, measured within hours in the peripheral blood, was dominated by an interferon transcriptional signature after influenza vaccination and by an inflammation signature after pneumococcal vaccination. Day 7 plasmablast responses induced by both vaccines was more pronounced after pneumococcal vaccination. Together, these results suggest that comparing global immune responses elicited by different vaccines will be critical to our understanding of the immune mechanisms underpinning successful vaccination.

Detrimental impacts of mixed-ion radiation on nervous system function.

Galactic cosmic radiation (GCR), composed of highly energetic and fully ionized atomic nuclei, produces diverse deleterious effects on the body. In researching the neurological risks of GCR exposures, including during human spaceflight, various ground-based single-ion GCR irradiation paradigms induce differential disruptions of cellular activity and overall behavior. However, it remains less clear how irradiation comprising a mix of multiple ions, more accurately recapitulating the space GCR environment, impacts the central nervous system. We therefore examined how mixed-ion GCR irradiation (two similar 5-6 beam combinations of protons, helium, oxygen, silicon and iron ions) influenced neuronal connectivity, functional generation of activity within neural circuits and cognitive behavior in mice. In electrophysiological recordings we find that space-relevant doses of mixed-ion GCR preferentially alter hippocampal inhibitory neurotransmission and produce related disruptions in the local field potentials of hippocampal oscillations. Such underlying perturbation in hippocampal network activity correspond with perturbed learning, memory and anxiety behavior.

Buildup and bistability in auditory streaming as an evidence accumulation process with saturation.

A repeating triplet-sequence ABA- of non-overlapping brief tones, A and B, is a valued paradigm for studying auditory stream formation and the cocktail party problem. The stimulus is "heard" either as a galloping pattern (integration) or as two interleaved streams (segregation); the initial percept is typically integration then followed by spontaneous alternations between segregation and integration, each being dominant for a few seconds. The probability of segregation grows over seconds, from near-zero to a steady value, defining the buildup function, BUF. Its stationary level increases with the difference in tone frequencies, DF, and the BUF rises faster. Percept durations have DF-dependent means and are gamma-like distributed. Behavioral and computational studies usually characterize triplet streaming either during alternations or during buildup. Here, our experimental design and modeling encompass both. We propose a pseudo-neuromechanistic model that incorporates spiking activity in primary auditory cortex, A1, as input and resolves perception along two network-layers downstream of A1. Our model is straightforward and intuitive. It describes the noisy accumulation of evidence against the current percept which generates switches when reaching a threshold. Accumulation can saturate either above or below threshold; if below, the switching dynamics resemble noise-induced transitions from an attractor state. Our model accounts quantitatively for three key features of data: the BUFs, mean durations, and normalized dominance duration distributions, at various DF values. It describes perceptual alternations without competition per se, and underscores that treating triplets in the sequence independently and averaging across trials, as implemented in earlier widely cited studies, is inadequate.

Temporal patterns of synchrony in a pyramidal-interneuron gamma (PING) network.

Synchronization in neural systems plays an important role in many brain functions. Synchronization in the gamma frequency band (30-100 Hz) is involved in a variety of cognitive phenomena; abnormalities of the gamma synchronization are found in schizophrenia and autism spectrum disorder. Frequently, the strength of synchronization is not high, and synchronization is intermittent even on short time scales (few cycles of oscillations). That is, the network exhibits intervals of synchronization followed by intervals of desynchronization. Neural circuit dynamics may show different distributions of desynchronization durations even if the synchronization strength is fixed. We use a conductance-based neural network exhibiting pyramidal-interneuron gamma rhythm to study the temporal patterning of synchronized neural oscillations. We found that changes in the synaptic strength (as well as changes in the membrane kinetics) can alter the temporal patterning of synchrony. Moreover, we found that the changes in the temporal pattern of synchrony may be independent of the changes in the average synchrony strength. Even though the temporal patterning may vary, there is a tendency for dynamics with short (although potentially numerous) desynchronizations, similar to what was observed in experimental studies of neural synchronization in the brain. Recent studies suggested that the short desynchronizations dynamics may facilitate the formation and the breakup of transient neural assemblies. Thus, the results of this study suggest that changes of synaptic strength may alter the temporal patterning of the gamma synchronization as to make the neural networks more efficient in the formation of neural assemblies and the facilitation of cognitive phenomena.

Ufd1 phosphorylation at serine 229 negatively regulates endoplasmic reticulum-associated degradation by inhibiting the interaction of Ufd1 with VCP.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through multistep processes termed ER-associated degradation (ERAD). Valosin-containing protein (VCP) plays a crucial role in ERAD as the interaction of ubiquitin fusion degradation protein 1 (Ufd1) with VCP via its SHP box motif (228F-S-G-S-G-N-R-L235) is required for ERAD. However, the mechanisms by which the VCP-Ufd1 interaction is regulated are not well understood. Here, we found that the serine 229 residue located in the Ufd1 SHP box is phosphorylated in vitro and in vivo by cyclic adenosine monophosphate-dependent protein kinase A (PKA), with this process being enhanced by either forskolin (an adenylyl cyclase activator) or calyculin A (a protein phosphatase inhibitor). Moreover, a phosphomimetic mutant (S229D) of Ufd1 as well as treatment by forskolin, calyculin A, or activated PKA strongly reduced Ufd1 binding affinity for VCP. Consistent with this, the Ufd1 S229D mutant significantly inhibited ERAD leading to the accumulation of ERAD substrates such as a tyrosinase mutant (C89R) and 3-hydroxy-3-methylglutaryl coenzyme A reductase. However, a non-phosphorylatable Ufd1 mutant (S229A) retained VCP-binding ability and was less effective in blocking ERAD. Collectively, our results support that Ufd1 S229 phosphorylation status mediated by PKA serves as a key regulatory point for the VCP-Ufd1 interaction and functional ERAD.

Histone deacetylase 6 inhibitor tubastatin A attenuates angiotensin II-induced hypertension by preventing cystathionine γ-lyase protein degradation.

Cystathionine γ-lyase (CSEγ) is a hydrogen sulfide (H2S)-producing enzyme. Endothelial H2S production can mediate vasodilatory effects, contributing to the alleviation of hypertension (high blood pressure). Recent studies have suggested a role of histone deacetylase 6 (HDAC6) in hypertension, although its underlying mechanisms are poorly understood. Here, we addressed the potential regulation of CSEγ by HDAC6 in angiotensin II (AngII)-induced hypertension and its molecular details focusing on CSEγ posttranslational modification. Treatment of mice with a selective HDAC6 inhibitor tubastatin A (TubA) alleviated high blood pressure and vasoconstriction induced by AngII. Cotreatment of the aorta and human aortic endothelial cells with TubA recovered AngII-mediated decreased H2S levels. AngII treatment upregulated HDAC6 mRNA and protein expression, but conversely downregulated CSEγ protein. Notably, potent HDAC6 inhibitors and HDAC6 siRNA as well as a proteasomal inhibitor increased CSEγ protein levels and blocked the downregulatory effect of AngII on CSEγ. In contrast, other HDAC isoforms-specific inhibitors and siRNAs did not show such blocking effects. Transfected CSEγ protein levels were also reciprocally regulated by AngII and TubA, and were reduced by wild-type, but not by deacetylase-deficient, HDAC6. Moreover, TubA significantly increased both protein stability and K73 acetylation level of CSEγ. Consistent with these results, AngII induced CSEγ ubiquitination and degradation, which was inhibited by TubA. Our results indicate that AngII promoted HDAC6-dependent deacetylation of CSEγ at K73 residue, leading to its ubiquitin-mediated proteolysis, which underlies AngII-induced hypertension. Overall, this study suggests that upregulation of CSEγ and H2S through HDAC6 inhibition may be considered as a valid strategy for preventing the progression of hypertension.

NEDD4-induced degradative ubiquitination of phosphatidylinositol 4-phosphate 5-kinase α and its implication in breast cancer cell proliferation.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family members generate phosphatidylinositol 4,5-bisphosphate (PIP2), a critical lipid regulator of diverse physiological processes. The PIP5K-dependent PIP2 generation can also act upstream of the oncogenic phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Many studies have demonstrated various mechanisms of spatiotemporal regulation of PIP5K catalytic activity. However, there are few studies on regulation of PIP5K protein stability. Here, we examined potential regulation of PIP5Kα, a PIP5K isoform, via ubiquitin-proteasome system, and its implication for breast cancer. Our results showed that the ubiquitin ligase NEDD4 (neural precursor cell expressed, developmentally down-regulated gene 4) mediated ubiquitination and proteasomal degradation of PIP5Kα, consequently reducing plasma membrane PIP2 level. NEDD4 interacted with the C-terminal region and ubiquitinated the N-terminal lysine 88 in PIP5Kα. In addition, PIP5Kα gene disruption inhibited epidermal growth factor (EGF)-induced Akt activation and caused significant proliferation defect in breast cancer cells. Notably, PIP5Kα K88R mutant that was resistant to NEDD4-mediated ubiquitination and degradation showed more potentiating effects on Akt activation by EGF and cell proliferation than wild-type PIP5Kα. Collectively, these results suggest that PIP5Kα is a novel degradative substrate of NEDD4 and that the PIP5Kα-dependent PIP2 pool contributing to breast cancer cell proliferation through PI3K/Akt activation is negatively controlled by NEDD4.

Neuroinvasive West Nile Infection Elicits Elevated and Atypically Polarized T Cell Responses That Promote a Pathogenic Outcome.

Most West Nile virus (WNV) infections are asymptomatic, but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death. Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection. However, other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage. We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen (HLA) class II tetramers. Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections. Independent of this, we also observed age related increases in WNV-specific T cell responses. Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells, whose presence was highly correlated with neuroinvasive disease. Moreover, a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects. More globally, subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections. Thus, subjects with neuroinvasive WNV infections exhibited elevated, dysregulated, and atypically polarized responses, suggesting that immune mediated damage may indeed contribute to pathogenic outcomes.

Autism-associated mutation inhibits protein kinase C-mediated neuroligin-4X enhancement of excitatory synapses.

Autism spectrum disorders (ASDs) comprise a highly heritable, multifarious group of neurodevelopmental disorders, which are characterized by repetitive behaviors and impairments in social interactions. Point mutations have been identified in X-linked Neuroligin (NLGN) 3 and 4X genes in patients with ASDs and all of these reside in their extracellular domains except for a single point mutation in the cytoplasmic domain of NLGN4X in which an arginine is mutated to a cysteine (R704C). Here we show that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707, and R704C completely eliminates T707 phosphorylation. Endogenous NLGN4X is intensely phosphorylated on T707 upon PKC stimulation in human neurons. Furthermore, a phospho-mimetic mutation at T707 has a profound effect on NLGN4X-mediated excitatory potentiation. Our results now establish an important interplay between a genetic mutation, a key posttranslational modification, and robust synaptic changes, which can provide insights into the synaptic dysfunction of ASDs.

Does volunteering decrease burnout? Healthcare professional and student perspectives on burnout and volunteering.

Background: Burnout among healthcare providers is a significant crisis in our healthcare system, especially in the context of the COVID-19 pandemic. The aim of this study was to understand what motivates healthcare workers and students to volunteer in their community as well as examine how volunteering relates to burnout. These findings can help health organizations better meet the needs of healthcare workers, as well as provide insights for non-profits that rely on volunteer professionals. Methods: Healthcare providers (N = 8), graduate healthcare students (N = 10), and undergraduate students (N = 14) who volunteered at community health fairs completed the OLBI burnout assessment and an individual semi-structured interview to characterize their attitudes toward volunteering and its relationship with burnout. Interviews were recorded, transcribed, and analyzed using a phenomenological approach, comparing themes across levels of burnout among providers and students. Results: Participants described that feeling burnt out decreased one's likelihood to volunteer, but also that volunteering prevented burnout. The OLBI scores showed that 79.2 and 20.8% of students were low and moderately burnt out respectively, and 87.5 and 12.5% of health professionals were low and moderately burnt out, respectively. Students volunteered for professional development while healthcare professionals cited a desire for a change in their day-to-day work as a reason to volunteer. Both students and health professionals often volunteered because they wanted to make a difference, it made them feel good, and/or they felt a responsibility to volunteer. COVID-19 had a wide range of effects on burnout and motivations to volunteer. Conclusion: Volunteering may be useful for preventing burnout among healthcare workers and students, but may not be helpful for those already experiencing burnout. Interview responses and the fact that none of the volunteers had high burnout levels according to their OLBI scores suggest those who choose to volunteer may be less burnt out. Healthcare organizations and schools can encourage volunteering by emphasizing the difference healthcare students and professionals can make through volunteering in the community. Increasing convenience and emphasizing professional development can help recruit and retain healthcare student volunteers. Highlighting the chance to diversify their scope of practice may help recruit and retain healthcare professional volunteers.

Acetylcholine receptor based chemogenetics engineered for neuronal inhibition and seizure control assessed in mice.

Epilepsy is a prevalent disorder involving neuronal network hyperexcitability, yet existing therapeutic strategies often fail to provide optimal patient outcomes. Chemogenetic approaches, where exogenous receptors are expressed in defined brain areas and specifically activated by selective agonists, are appealing methods to constrain overactive neuronal activity. We developed BARNI (Bradanicline- and Acetylcholine-activated Receptor for Neuronal Inhibition), an engineered channel comprised of the α7 nicotinic acetylcholine receptor ligand-binding domain coupled to an α1 glycine receptor anion pore domain. Here we demonstrate that BARNI activation by the clinical stage α7 nicotinic acetylcholine receptor-selective agonist bradanicline effectively suppressed targeted neuronal activity, and controlled both acute and chronic seizures in male mice. Our results provide evidence for the use of an inhibitory acetylcholine-based engineered channel activatable by both exogenous and endogenous agonists as a potential therapeutic approach to treating epilepsy.

The fasciola cinereum of the hippocampal tail as an interventional target in epilepsy.

Targeted tissue ablation involving the anterior hippocampus is the standard of care for patients with drug-resistant mesial temporal lobe epilepsy. However, a substantial proportion continues to suffer from seizures even after surgery. We identified the fasciola cinereum (FC) neurons of the posterior hippocampal tail as an important seizure node in both mice and humans with epilepsy. Genetically defined FC neurons were highly active during spontaneous seizures in epileptic mice, and closed-loop optogenetic inhibition of these neurons potently reduced seizure duration. Furthermore, we specifically targeted and found the prominent involvement of FC during seizures in a cohort of six patients with epilepsy. In particular, targeted lesioning of the FC in a patient reduced the seizure burden present after ablation of anterior mesial temporal structures. Thus, the FC may be a promising interventional target in epilepsy.

Genetic alterations in peritoneal metastatic tumors predicted the outcomes for hyperthermic intraperitoneal chemotherapy.

Introduction: Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) are considered for patients with peritoneal metastasis (PM). However, patients selection that relies on conventional prognostic factors is not yet optimal. In this study, we performed whole exome sequencing (WES) to establish tumor molecular characteristics and expect to identify prognosis profiles for PM management. Methods: In this study, blood and tumor samples were collected from patients with PM before HIPEC. Tumor molecular signatures were determined using WES. Patient cohort was divided into responders and non-responders according to 12-month progression-free survival (PFS). Genomic characteristics between the two cohorts were compared to study potential targets. Results: In total, 15 patients with PM were enrolled in this study. Driver genes and enriched pathways were identified from WES results. AGAP5 mutation was found in all responders. This mutation was significantly associated with better OS (p = 0.00652). Conclusions: We identified prognostic markers that might be useful to facilitate decision-making before CRS/HIPEC.

Is nature relatedness associated with better mental health? An exploratory study on Vietnamese university students.

OBJECTIVE: This study aims to examine the relationship between nature relatedness (NR) on subjective positive well-being (life satisfaction) and mental disorders (depression, stress, and anxiety) in Vietnamese university students. PARTICIPANTS: 340 Hue University students voluntarily took part in the survey. METHODS: The Nature Relatedness Scale, Depression, Anxiety, and Stress Scale and Satisfaction with Life Scale were used. Bivariate correlation, single regression, and moderation analysis were conducted to investigate the relationship between NR and the mental health of university students and the moderating role of NR. RESULTS: Higher levels of NR significantly predicted higher levels of life satisfaction but did not significantly predict fewer symptoms of depression, anxiety, and stress. NR significantly moderated the link between life satisfaction and depression, anxiety, and stress. CONCLUSIONS: The findings of this study imply that efforts to enhance students' mental health should include efforts to promote their NR.

Inflammatory bone marrow signaling in pediatric acute myeloid leukemia distinguishes patients with poor outcomes.

High levels of the inflammatory cytokine IL-6 in the bone marrow are associated with poor outcomes in pediatric acute myeloid leukemia (pAML), but its etiology remains unknown. Using RNA-seq data from pre-treatment bone marrows of 1489 children with pAML, we show that > 20% of patients have concurrent IL-6, IL-1, IFNα/ß, and TNFα signaling activity and poorer outcomes. Targeted sequencing of pre-treatment bone marrow samples from affected patients (n = 181) revealed 5 highly recurrent patterns of somatic mutation. Using differential expression analyses of the most common genomic subtypes (~60% of total), we identify high expression of multiple potential drivers of inflammation-related treatment resistance. Regardless of genomic subtype, we show that JAK1/2 inhibition reduces receptor-mediated inflammatory signaling by leukemic cells in-vitro. The large number of high-risk pAML genomic subtypes presents an obstacle to the development of mutation-specific therapies. Our findings suggest that therapies targeting inflammatory signaling may be effective across multiple genomic subtypes of pAML.

Deep brain optogenetics without intracranial surgery.

Achieving temporally precise, noninvasive control over specific neural cell types in the deep brain would advance the study of nervous system function. Here we use the potent channelrhodopsin ChRmine to achieve transcranial photoactivation of defined neural circuits, including midbrain and brainstem structures, at unprecedented depths of up to 7 mm with millisecond precision. Using systemic viral delivery of ChRmine, we demonstrate behavioral modulation without surgery, enabling implant-free deep brain optogenetics.