19F-NMR Probing of Ion-Induced Conformational Changes in Detergent-Solubilized and Nanodisc-Reconstituted NCX_Mj.

Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.

Single-cell multi-ome and immune profiles of the Inspiration4 crew reveal conserved, cell-type, and sex-specific responses to spaceflight.

Spaceflight induces an immune response in astronauts. To better characterize this effect, we generated single-cell, multi-ome, cell-free RNA (cfRNA), biochemical, and hematology data for the SpaceX Inspiration4 (I4) mission crew. We found that 18 cytokines/chemokines related to inflammation, aging, and muscle homeostasis changed after spaceflight. In I4 single-cell multi-omics data, we identified a "spaceflight signature" of gene expression characterized by enrichment in oxidative phosphorylation, UV response, immune function, and TCF21 pathways. We confirmed the presence of this signature in independent datasets, including the NASA Twins Study, the I4 skin spatial transcriptomics, and 817 NASA GeneLab mouse transcriptomes. Finally, we observed that (1) T cells showed an up-regulation of FOXP3, (2) MHC class I genes exhibited long-term suppression, and (3) infection-related immune pathways were associated with microbiome shifts. In summary, this study reveals conserved and distinct immune disruptions occurring and details a roadmap for potential countermeasures to preserve astronaut health.

Single-cell analysis identifies conserved features of immune dysfunction in simulated microgravity and spaceflight.

Microgravity is associated with immunological dysfunction, though the mechanisms are poorly understood. Here, using single-cell analysis of human peripheral blood mononuclear cells (PBMCs) exposed to short term (25 hours) simulated microgravity, we characterize altered genes and pathways at basal and stimulated states with a Toll-like Receptor-7/8 agonist. We validate single-cell analysis by RNA sequencing and super-resolution microscopy, and against data from the Inspiration-4 (I4) mission, JAXA (Cell-Free Epigenome) mission, Twins study, and spleens from mice on the International Space Station. Overall, microgravity alters specific pathways for optimal immunity, including the cytoskeleton, interferon signaling, pyroptosis, temperature-shock, innate inflammation (e.g., Coronavirus pathogenesis pathway and IL-6 signaling), nuclear receptors, and sirtuin signaling. Microgravity directs monocyte inflammatory parameters, and impairs T cell and NK cell functionality. Using machine learning, we identify numerous compounds linking microgravity to immune cell transcription, and demonstrate that the flavonol, quercetin, can reverse most abnormal pathways. These results define immune cell alterations in microgravity, and provide opportunities for countermeasures to maintain normal immunity in space.

Overlapping Stevens-Johnson Syndrome and DRESS Syndrome Caused by Phenobarbital: A Vietnamese Case Report.

Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) Syndrome and Stevens-Johnson Syndrome (SJS) are severe cutaneous adverse reactions to drugs. Those reactions which are rare in children can be especially severe and challenging to diagnose and manage. Herein we present a 59-month-old male who presented with a rash, fever, and multiple organ dysfunction initiation of Phenobarbital for epilepsy. Diagnosis of ovelaping SJS and DRESS syndrome had been made based on clinical manifestations accompanied with skin biopsy according to RegisSCAR diagnostic criteria. A therapy with intravenous immune globulin (IVIG), corticosteroids and supportive care was given successfully for the patient. This case underscored the significance of promptly and effectively recognizing and managing these intricate reactions.

Recent advances in uveitis therapy: focus on selected phase 2 and 3 clinical trials.

INTRODUCTION: Uveitis is a heterogeneous group of ocular conditions characterized by inflammation of the uveal tract. It is a leading cause of blindness in developed countries and exerts significant psychological, social, and economic impact on both patients and the larger society. While there are numerous pharmacotherapy options, posterior segment noninfectious uveitis remains a significant challenge to treat due to its severity, chronicity, and high recurrence rates. AREAS COVERED: The index review highlights the unmet needs of uveitis pharmacotherapy and its research and the shortcomings of existing ocular and systemic therapeutic options for noninfectious uveitis. The more promising novel ocular drug delivery methods and therapeutic targets/drugs are discussed, and evidence from the clinical trials is evaluated. EXPERT OPINION: There has been incredible growth in the number of treatment options available to uveitis patients today, especially with the new generation of biologic drugs. Available evidence suggests that these newer options may be superior to conventional immunosuppressive therapies in terms of efficacy and side effect profiles. Further high-quality research and additional clinical trials will be needed to clarify their roles in the stepladder treatment approach of noninfectious uveitis.

Synthesis of a BC-Dihydrodipyrrin Building Block of Bacteriochlorophyll a.

A strategy for the synthesis of bacteriochlorophyll a relies on joining AD and BC halves that contain the requisite stereochemical configurations of the target macrocycle. The BC half (1) is a dihydrodipyrrin bearing a dimethoxymethyl group at the 1-position, a ß-ketoester at the 8-position, and (R)-2-methyl and (R)-3-ethyl substituents in the pyrroline ring. An established route to AD-dihydrodipyrrins (Pd-mediated coupling of a 2-halopyrrole with a chiral 4-pentynoic acid followed by Petasis methenylation, acidic hydrolysis, Paal-Knorr ring closure, and Riley oxidation) proved to be unviable for BC-dihydrodipyrrins given the presence of the ß-ketoester unit. A route presented here entails Pd-mediated coupling of a 2-halopyrrole (2) with (3R,4R)-4-ethyl-1,1-dimethoxy-3-methylhex-5-yn-2-one (3), anti-Markovnikov hydration of the alkyne to give the 1,4-diketone, and Paal-Knorr ring closure. Compound 3 was prepared by Schreiber-modified Nicholas reaction beginning with (S)-4-isopropyl-3-propionyloxazolidin-2-one and the hexacarbonyldicobalt complex of (±) 3-methoxy-1-(trimethylsilyl)pentyne followed by transformation of the aldehyde derived therefrom to the 1,1-dimethoxymethylcarbonyl motif. The absolute stereochemical configuration of the Schreiber-Nicholas alkylation product was confirmed by single-crystal X-ray diffraction, whereas the BC half (1) by 1H NMR spectroscopy showed a J value of 2.9 Hz consistent with the trans-configuration. Taken together, the route provides a key chiral building block for the synthesis of photosynthetic tetrapyrroles and analogues.

Mutations to the BTN2A1 Linker Region Impact Its Homodimerization and Its Cytoplasmic Interaction with Phospho-Antigen-Bound BTN3A1.

Intracellular binding of small-molecule phospho-Ags to the HMBPP receptor complex in infected cells leads to extracellular detection by T cells expressing the Vγ9Vδ2 TCR, a noncanonical method of Ag detection. The butyrophilin proteins BTN2A1 and BTN3A1 are part of the complex; however, their precise roles are unclear. We suspected that BTN2A1 and BTN3A1 form a tetrameric (dimer of dimers) structure, and we wanted to probe the importance of the BTN2A1 homodimer. We analyzed mutations to human BTN2A1, using internal domain or full-length BTN2A1 constructs, expressed in Escherichia coli or human K562 cells, that might disrupt its structure and/or function. Although BTN2A1 is a disulfide-linked homodimer, mutation of cysteine residues C247 and C265 did not affect the ability to stimulate T cell IFN-γ production by ELISA. Two mutations of the juxtamembrane region (at EKE282) failed to impact BTN2A1 function. In contrast, single point mutations (L318G and L325G) near the BTN2A1 B30.2 domain blocked phospho-Ag response. Size exclusion chromatography and nuclear magnetic resonance (NMR) experiments showed that the isolated BTN2A1 B30.2 domain is a homodimer, even in the absence of its extracellular and transmembrane region. [31P]-NMR experiments confirmed that HMBPP binds to BTN3A1 but not BTN2A1, and binding abrogates signals from both phosphorus atoms. Furthermore, the BTN2A1 L325G mutation but not the L318G mutation prevents both homodimerization of BTN2A1 internal domain constructs in size exclusion chromatography (and NMR) experiments and their binding to HMBPP-bound BTN3A1 in isothermal titration calorimetry experiments. Together, these findings support the importance of homodimerization within the BTN2A1 internal domain for phospho-Ag detection.

Four Routes to 3-(3-Methoxy-1,3-dioxopropyl)pyrrole, a Core Motif of Rings C and E in Photosynthetic Tetrapyrroles.

The photosynthetic tetrapyrroles share a common structural feature comprised of a ß-ketoester motif embedded in an exocyclic ring (ring E). As part of a total synthesis program aimed at preparing native structures and analogues, 3-(3-methoxy-1,3-dioxopropyl)pyrrole was sought. The pyrrole is a precursor to analogues of ring C and the external framework of ring E. Four routes were developed. Routes 1-3 entail a Pd-mediated coupling process of a 3-iodopyrrole with potassium methyl malonate, whereas route 4 relies on electrophilic substitution of TIPS-pyrrole with methyl malonyl chloride. Together, the four routes afford considerable latitude. A long-term objective is to gain the capacity to create chlorophylls and bacteriochlorophylls and analogues thereof by facile de novo means for diverse studies across the photosynthetic sciences.

B Cells Promote T Cell Immunosenescence and Mammalian Aging Parameters.

A dysregulated adaptive immune system is a key feature of aging, and is associated with age-related chronic diseases and mortality. Most notably, aging is linked to a loss in the diversity of the T cell repertoire and expansion of activated inflammatory age-related T cell subsets, though the main drivers of these processes are largely unknown. Here, we find that T cell aging is directly influenced by B cells. Using multiple models of B cell manipulation and single-cell omics, we find B cells to be a major cell type that is largely responsible for the age-related reduction of naive T cells, their associated differentiation towards pathogenic immunosenescent T cell subsets, and for the clonal restriction of their T cell receptor (TCR). Accordingly, we find that these pathogenic shifts can be therapeutically targeted via CD20 monoclonal antibody treatment. Mechanistically, we uncover a new role for insulin receptor signaling in influencing age-related B cell pathogenicity that in turn induces T cell dysfunction and a decline in healthspan parameters. These results establish B cells as a pivotal force contributing to age-associated adaptive immune dysfunction and healthspan outcomes, and suggest new modalities to manage aging and related multi-morbidity.

Age-related brain atrophy is not a homogenous process: Different functional brain networks associate differentially with aging and blood factors.

Aging is characterized by a progressive loss of brain volume at an estimated rate of 5% per decade after age 40. While these morphometric changes, especially those affecting gray matter and atrophy of the temporal lobe, are predictors of cognitive performance, the strong association with aging obscures the potential parallel, but more specific role, of individual subject physiology. Here, we studied a cohort of 554 human subjects who were monitored using structural MRI scans and blood immune protein concentrations. Using machine learning, we derived a cytokine clock (CyClo), which predicted age with good accuracy (Mean Absolute Error = 6 y) based on the expression of a subset of immune proteins. These proteins included, among others, Placenta Growth Factor (PLGF) and Vascular Endothelial Growth Factor (VEGF), both involved in angiogenesis, the chemoattractant vascular cell adhesion molecule 1 (VCAM-1), the canonical inflammatory proteins interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα), the chemoattractant IP-10 (CXCL10), and eotaxin-1 (CCL11), previously involved in brain disorders. Age, sex, and the CyClo were independently associated with different functionally defined cortical networks in the brain. While age was mostly correlated with changes in the somatomotor system, sex was associated with variability in the frontoparietal, ventral attention, and visual networks. Significant canonical correlation was observed for the CyClo and the default mode, limbic, and dorsal attention networks, indicating that immune circulating proteins preferentially affect brain processes such as focused attention, emotion, memory, response to social stress, internal evaluation, and access to consciousness. Thus, we identified immune biomarkers of brain aging which could be potential therapeutic targets for the prevention of age-related cognitive decline.

Ligand-induced interactions between butyrophilin 2A1 and 3A1 internal domains in the HMBPP receptor complex.

The ligand-bound (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) receptor (BTN3A1 and BTN2A1) is detectable by the T cell receptor (TCR) of Vγ9Vδ2 T cells. Although BTN3A1 binds to phosphoantigens (pAgs), the mechanisms resulting in receptor activation are not clear. We used CRISPR-Cas9, ELISA, nano-bioluminescence resonance energy transfer (BRET), and isothermal titration calorimetry (ITC) to evaluate the role of BTN2A1. Depletion of BTN2A1 and rescue experiments demonstrate that its internal domain is essential for pAg detection. Internal hetero-BRET signals are observed between BTN2A1 and BTN3A1 that are increased by pAg. ITC detects a direct interaction between the intracellular domains of BTN3A1 and BTN2A1 only in the presence of pAg. This interaction is abrogated by removal of the BTN2A1 juxtamembrane (JM) region but not by removal of the BTN3A1 JM region. Regional mutations between BTN2A1 316-326 clearly affect the interferon γ (IFNγ) response and hetero-BRET signal. Mutations to amino acids L318, W320, and L325 indicate that these amino acids are crucial. This study demonstrates a pAg-inducible interaction between BTN2A1 and BTN3A1 internal domains.

An inflammatory aging clock (iAge) based on deep learning tracks multimorbidity, immunosenescence, frailty and cardiovascular aging.

While many diseases of aging have been linked to the immunological system, immune metrics capable of identifying the most at-risk individuals are lacking. From the blood immunome of 1,001 individuals aged 8-96 years, we developed a deep-learning method based on patterns of systemic age-related inflammation. The resulting inflammatory clock of aging (iAge) tracked with multimorbidity, immunosenescence, frailty and cardiovascular aging, and is also associated with exceptional longevity in centenarians. The strongest contributor to iAge was the chemokine CXCL9, which was involved in cardiac aging, adverse cardiac remodeling and poor vascular function. Furthermore, aging endothelial cells in human and mice show loss of function, cellular senescence and hallmark phenotypes of arterial stiffness, all of which are reversed by silencing CXCL9. In conclusion, we identify a key role of CXCL9 in age-related chronic inflammation and derive a metric for multimorbidity that can be utilized for the early detection of age-related clinical phenotypes.

Survival in resectable pancreatic ductal adenocarcinoma with para-aortic lymph node dissection: A retrospective study in Vietnamese population.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a high recurrence rate and poor outcome. Lymph node (LN) metastasis, especially para-aortic LN (PALN), is an important prognostic factor. PALN assessment through sampling with frozen-section analysis is a validated method. Our aim was to evaluate the prognostic impact of PALN on overall survival (OS) in patients who underwent standard pancreaticoduodenectomy, lymphadenectomy with PALN sampling, as well as to identify other prognostic factors for survival. METHODS: Our retrospective study included 89 PDAC patients undergoing radical resection with PALN sampling. The patients were classified into PALN(+) (n = 11) and PALN(-) (n = 78). Univariate and multivariate analyses of 1-year and 3-year OS and Kaplan-Meier model were used. RESULTS: OS after 1-year for PALN(+) and PALN(-) was 18.2 and 56.4%, after 3-year was 15.4% and 0%, respectively. Tumor differentiation, LN metastasis (LN(-), LN(+) PALN(-), LN(+) PALN(+)) were significant prognostic factors in both univariate and multivariate analyses for 1-year OS, and neural invasion (PN) was the solely significant factor for 3-year OS (p < 0.05). Kaplan-Meier estimate showed that OS of PALN(+) and PN (+) was significantly lower than the negative group, respectively (p < 0.05). No statistical difference in OS was seen between LN(-) and LN(+) PALN(-); and between LN(+) PALN(-) and PALN(+) (p = 0.107). Patients with PN (-) PALN(+) had similar OS compared to PN (+) PALN(-) (p > 0.05). CONCLUSION: PDAC had a poor outcome despite treatment with radical resection. Further follow-up should be conducted to determine the role of surgery in PALN(+)and PN invasion.

Asymmetric Synthesis of a Bacteriochlorophyll Model Compound Containing trans-Dialkyl Substituents in Ring D.

Challenges to the de novo synthesis of bacteriochlorophyll a (BChl a), the chief pigment for anoxygenic bacterial photosynthesis, include creating the macrocycle along with the trans-dialkyl substituents in both pyrroline rings (B and D). A known route to a model bacteriochlorophyll with a gem-dimethyl group in each pyrroline ring has been probed for utility in the synthesis of BChl a by preparation of a hybrid macrocycle (BC-1), which contains a trans-dialkyl group in ring D and a gem-dimethyl group in ring B. Stereochemical definition began with the synthesis of (2S,3S)-2-ethyl-3-methylpent-4-ynoic acid, a precursor to the trans-dialkyl-substituted AD dihydrodipyrrin. Knoevenagel condensation of the latter and a gem-dimethyl, ß-ketoester-substituted BC dihydrodipyrrin afforded the enone (E, 70%; Z, 3%); subsequent double-ring cyclization of the E-enone (via Nazarov, electrophilic aromatic substitution, and elimination reactions) gave BC-1 (53% yield) along with a trace of chlorin byproduct (1.4% relative to BC-1 upon fluorescence assay). BC-1 exhibited the desired trans-dialkyl stereochemistry in ring D and was obtained as a 7:1 mixture of (expected) epimers owing to the configuration of the 132-carbomethoxy substituent. The strategy wherein trans-dialkyl substituents are installed very early and carried through to completion, as validated herein, potentially opens a synthetic path to native photosynthetic pigments.

A Robust Method Uncovers Significant Context-Specific Heritability in Diverse Complex Traits.

Gene-environment interactions (GxE) can be fundamental in applications ranging from functional genomics to precision medicine and is a conjectured source of substantial heritability. However, unbiased methods to profile GxE genome-wide are nascent and, as we show, cannot accommodate general environment variables, modest sample sizes, heterogeneous noise, and binary traits. To address this gap, we propose a simple, unifying mixed model for gene-environment interaction (GxEMM). In simulations and theory, we show that GxEMM can dramatically improve estimates and eliminate false positives when the assumptions of existing methods fail. We apply GxEMM to a range of human and model organism datasets and find broad evidence of context-specific genetic effects, including GxSex, GxAdversity, and GxDisease interactions across thousands of clinical and molecular phenotypes. Overall, GxEMM is broadly applicable for testing and quantifying polygenic interactions, which can be useful for explaining heritability and invaluable for determining biologically relevant environments.

Synthesis of the Ring C Pyrrole of Native Chlorophylls and Bacteriochlorophylls.

As part of a program to develop practical syntheses of members of the family of (bacterio)chlorophylls, two routes to 2-iodo-3-methyl-4-(3-methoxy-1,3-dioxopropyl)pyrrole, a precursor of the universal ring C, have been developed. The ß-ketoester of ring C is expected to give rise to ring E upon Knoevenagel condensation and Nazarov cyclization with a ring D constituent as demonstrated in an analogue synthesis. Two viable routes were developed beginning with N-TIPS-pyrrole or with 4-oxo-2-pentene and TosMIC, affording multi-gram-quantities of this ostensibly simple pyrrole.

Hypervalent Iodine Reagent Mediated Oxidative Heterocyclization of Aldoximes with Heterocyclic Alkenes.

An efficient cycloaddition of heterocyclic alkenes with nitrile oxides generated in situ from the corresponding aldoximes using organohypervalent iodine(III) reagent, [hydroxy(tosyloxy)iodo]benzene (Koser's reagent), has been developed. The oxidative cyclization of various aldoximes with 1-propene-1,3-sultone affords the respective isoxazoline-ring-fused heterobicyclic products in moderate to good yields. Furthermore, the reaction of aldoxime with a cyclic phospholene-oxide under similar conditions produces the corresponding heterobicyclic phospholene oxides in moderate yields. The structures of bicyclic phospholene oxide and two sultones were established by single-crystal X-ray crystallography.

The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

Small isoprenoid diphosphates, such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9Vδ2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (ST296AA or T304A) investigated, confirm that the backbone amide of at least one Thr (Thr304), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr296/297) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of Vγ9Vδ2 T cells by HMBPP, zoledronate, and POM2-C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.-Nguyen, K., Li, J., Puthenveetil, R., Lin, X., Poe, M. M., Hsiao, C.-H. C., Vinogradova, O., Wiemer, A. J. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

Nanodiscs and Solution NMR: preparation, application and challenges.

Nanodiscs provide an excellent system for the structure-function investigation of membrane proteins. Its direct advantage lies in presenting a water soluble form of an otherwise hydrophobic molecule, making it amenable to a plethora of solution techniques. Nuclear Magnetic Resonance is one such high resolution approach that looks at the structure and dynamics of a protein with atomic level precision. Recently, there has been a breakthrough in making nanodiscs more susceptible for structure determination by solution NMR, yet it still remains to become the preferred choice for a membrane mimetic. In this practical review, we provide a general discourse on nanodisc and its application to solution NMR. We also offer potential solutions to remediate the technical challenges associated with nanodisc preparation and the choice of proper experimental set-ups. Along with discussing several structural applications, we demonstrate an alternative use of nanodiscs for functional studies, where we investigated the phosphorylation of a cell surface receptor, Integrin. This is the first successful manifestation of observing activated receptor phosphorylation in nanodiscs through NMR. We additionally present an on-column method for nanodisc preparation with multiple strategies and discuss the potential use of alternative nanoscale phospholipid bilayer systems like SMA lipid discs and Saposin-A lipoprotein discs.