STING controls T cell memory fitness during infection through T cell-intrinsic and IDO-dependent mechanisms.

Stimulator of interferon genes (STING) signaling has been extensively studied in inflammatory diseases and cancer, while its role in T cell responses to infection is unclear. Using Listeria monocytogenes strains engineered to induce different levels of c-di-AMP, we found that high STING signals impaired T cell memory upon infection via increased Bim levels and apoptosis. Unexpectedly, reduction of TCR signal strength or T cell-STING expression decreased Bim expression, T cell apoptosis, and recovered T cell memory. We found that TCR signal intensity coupled STING signal strength to the unfolded protein response (UPR) and T cell survival. Under strong STING signaling, Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition also reduced apoptosis and led to a recovery of T cell memory in STING sufficient CD8 T cells. Thus, STING signaling regulates CD8 T cell memory fitness through both cell-intrinsic and extrinsic mechanisms. These studies provide insight into how IDO and STING therapies could improve long-term T cell protective immunity.

Multicenter, Randomized Trial of a Bionic Pancreas in Type 1 Diabetes.

BACKGROUND: Currently available semiautomated insulin-delivery systems require individualized insulin regimens for the initialization of therapy and meal doses based on carbohydrate counting for routine operation. In contrast, the bionic pancreas is initialized only on the basis of body weight, makes all dose decisions and delivers insulin autonomously, and uses meal announcements without carbohydrate counting. METHODS: In this 13-week, multicenter, randomized trial, we randomly assigned in a 2:1 ratio persons at least 6 years of age with type 1 diabetes either to receive bionic pancreas treatment with insulin aspart or insulin lispro or to receive standard care (defined as any insulin-delivery method with unblinded, real-time continuous glucose monitoring). The primary outcome was the glycated hemoglobin level at 13 weeks. The key secondary outcome was the percentage of time that the glucose level as assessed by continuous glucose monitoring was below 54 mg per deciliter; the prespecified noninferiority limit for this outcome was 1 percentage point. Safety was also assessed. RESULTS: A total of 219 participants 6 to 79 years of age were assigned to the bionic-pancreas group, and 107 to the standard-care group. The glycated hemoglobin level decreased from 7.9% to 7.3% in the bionic-pancreas group and did not change (was at 7.7% at both time points) in the standard-care group (mean adjusted difference at 13 weeks, -0.5 percentage points; 95% confidence interval [CI], -0.6 to -0.3; P<0.001). The percentage of time that the glucose level as assessed by continuous glucose monitoring was below 54 mg per deciliter did not differ significantly between the two groups (13-week adjusted difference, 0.0 percentage points; 95% CI, -0.1 to 0.04; P<0.001 for noninferiority). The rate of severe hypoglycemia was 17.7 events per 100 participant-years in the bionic-pancreas group and 10.8 events per 100 participant-years in the standard-care group (P = 0.39). No episodes of diabetic ketoacidosis occurred in either group. CONCLUSIONS: In this 13-week, randomized trial involving adults and children with type 1 diabetes, use of a bionic pancreas was associated with a greater reduction than standard care in the glycated hemoglobin level. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others; ClinicalTrials.gov number, NCT04200313.).

Female Gene Networks Are Expressed in Myofibroblast-Like Smooth Muscle Cells in Vulnerable Atherosclerotic Plaques.

BACKGROUND: Women presenting with coronary artery disease more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. METHODS: Gene regulatory networks were created using RNAseq gene expression data from human carotid atherosclerotic plaques. The networks were prioritized based on sex bias, relevance for smooth muscle biology, and coronary artery disease genetic enrichment. Network expression was linked to histologically determined plaque phenotypes. In addition, their expression in plaque cell types was studied at single-cell resolution using single-cell RNAseq. Finally, their relevance for disease progression was studied in female and male Apoe-/- mice fed a Western diet for 18 and 30 weeks. RESULTS: Here, we identify multiple sex-stratified gene regulatory networks from human carotid atherosclerotic plaques. Prioritization of the female networks identified 2 main SMC gene regulatory networks in late-stage atherosclerosis. Single-cell RNA sequencing mapped these female networks to 2 SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like network was mostly expressed in plaques that were vulnerable in women. Finally, the mice ortholog of key driver gene MFGE8 (milk fat globule EGF and factor V/VIII domain containing) showed retained expression in advanced plaques from female mice but was downregulated in male mice during atherosclerosis progression. CONCLUSIONS: Female atherosclerosis is characterized by gene regulatory networks that are active in fibrous vulnerable plaques rich in myofibroblast-like SMCs.

Integrating transition readiness assessment into clinical practice: Adaptation of the UNC TRXANSITION index into the Cerner electronic medical record.

PURPOSE: To describe the process of developing, and evaluating the feasibility and acceptability of, an EMR-based transition readiness assessment. DESIGN AND METHODS: A Cerner-based version of the UNC TRxANSITION Index was implemented across four pediatric subspecialty clinics: epilepsy, inflammatory bowel disease; type 1 diabetes, oncology survivorship. The feasibility was assessed by each's clinic's ability to meet form completion goals and their assessment rate. Acceptability was assessed via family refusal rate, a staff-completed feedback questionnaire, and whether the form was adopted into routine clinical care after completion of the pilot study. RESULTS: All clinics met form completion goals (N = 10/clinic). The assessment rate ranged from 66 to 100% across clinics. No families refused completion of the form. Most staff (70%) reported completing the form in <10 min. Staff reported on challenges experienced and provided recommendations to streamline administration and enhance clinical care. All staff reported the form helped them identify knowledge gaps in their patients. Two clinics continued using the form following completion of the pilot study. CONCLUSIONS: Implementation was most feasible in clinics that were well-staffed and had lengthier patient visits, however, time and staff resources were the biggest challenges to implementation across clinics. Based on staff feedback to improve efficiency and developmentally-tailor assessment, the form will be divided into Beginner Skills and Advanced Skills. PRACTICAL IMPLICATIONS: Integrating transition readiness assessment into the EMR has the potential to improve clinical care by facilitating staff's ability to efficiently identify knowledge gaps in their transition-aged patients and intervene.

Safety of belimumab in adult patients with systemic lupus erythematosus: Results of a large integrated analysis of controlled clinical trial data.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a chronic, autoimmune disease that affects multiple organ systems. Belimumab, a targeted human monoclonal antibody, binds to and inhibits soluble B-lymphocyte stimulator. The safety and efficacy of belimumab has consistently been demonstrated in multiple clinical trials for the treatment of patients with active SLE. Integration of these data provides an additional opportunity to explore the safety of belimumab in a larger and more diverse population. This post hoc pooled analysis of clinical studies evaluated the safety profile of belimumab versus placebo in adults with SLE. METHODS: This was a pooled post hoc analysis of 52-week safety data from one Phase 2 and five Phase 3 belimumab trials in adult patients with SLE. Patients received ≥1 dose of placebo or belimumab (1, 4, or 10 mg/kg intravenous or 200 mg subcutaneous), plus standard therapy. Outcomes included the incidence of adverse events (AEs), serious AEs (SAEs), severe AEs, AEs of special interest (AESI), and mortality. RESULTS: Across 4170 patients (placebo: N = 1355; belimumab: N = 2815), baseline demographics, disease characteristics, and treatment exposure were similar for placebo and belimumab. Most patients (placebo: 76.6%; belimumab: 81.0%) completed the protocol Week 52 visit. Overall, incidence of AEs, SAEs, severe AEs, AESI, and mortality were similar between groups. In both groups, the most commonly reported SAEs by system organ class were infections and infestations (placebo: 5.9%; belimumab: 5.4%) and renal and urinary disorders (placebo: 2.2%; belimumab: 1.7%). Additionally, a greater proportion of patients experienced AESI with belimumab versus placebo for post-infusion/injection systemic reactions (placebo: 8.1%; belimumab: 10.2%). Mortality rates were similar between groups (placebo: 0.4%; belimumab: 0.6%). CONCLUSIONS: These results are consistent with those of the individual studies, BASE, BLISS-LN, and long-term extension studies, making belimumab one of the most studied SLE treatments for safety. Collectively, this evidence continues to support a positive benefit-risk profile of belimumab in the treatment of adult patients with SLE.

Comorbidities increase COVID-19 hospitalization in young people with type 1 diabetes.

OBJECTIVES: We evaluated COVID-19 outcomes in children and young adults with type 1 diabetes (T1D) to determine if those with comorbidities are more likely to experience severe COVID-19 compared to those without. RESEARCH DESIGN AND METHODS: This cross-sectional study included questionnaire data on patients <25 years of age with established T1D and laboratory-confirmed COVID-19 from 52 sites across the US between April 2020 and October 2021. We examined patient factors and COVID-19 outcomes between those with and without comorbidities. Multivariate logistic regression analysis examined the odds of hospitalization among groups, adjusting for age, HbA1c, race and ethnicity, insurance type and duration of diabetes. RESULTS: Six hundred fifty-one individuals with T1D and COVID-19 were analyzed with mean age 15.8 (SD 4.1) years. At least one comorbidity was present in 31%, and more than one in 10%. Obesity and asthma were the most frequently reported comorbidities, present in 19% and 17%, respectively. Hospitalization occurred in 17% of patients and 52% of hospitalized patients required ICU level care. Patients with at least one comorbidity were almost twice as likely to be hospitalized with COVID-19 than patients with no comorbidities (Odds ratio 2.0, 95% CI: 1.3-3.1). This relationship persisted after adjusting for age, HbA1c, race and ethnicity (minority vs nonminority), insurance type (public vs. private), and duration of diabetes. CONCLUSIONS: Our findings show that comorbidities increase the risk for hospitalization with COVID-19 in children and young adults highlighting the need for tailored COVID-19 prevention and treatment strategies in T1D.

Ramified rolling circle amplification for synthesis of nucleosomal DNA sequences.

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that ramified rolling circle amplification (RCA) can be used to produce milligram amounts of a genomic nucleosomal DNA fragment in a scalable, one-pot reaction overnight. The protocol is efficient and flexible in choice of DNA sequence. It yields 10-fold more product than PCR, and rivals production using plasmids. We demonstrate the approach by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.

NFκB-Pim-1-Eomesodermin axis is critical for maintaining CD8 T-cell memory quality.

T-cell memory is critical for long-term immunity. However, the factors involved in maintaining the persistence, function, and phenotype of the memory pool are undefined. Eomesodermin (Eomes) is required for the establishment of the memory pool. Here, we show that in T cells transitioning to memory, the expression of high levels of Eomes is not constitutive but rather requires a continuum of cell-intrinsic NFκB signaling. Failure to maintain NFκB signals after the peak of the response led to impaired Eomes expression and a defect in the maintenance of CD8 T-cell memory. Strikingly, we found that antigen receptor [T-cell receptor (TCR)] signaling regulates this process through expression of the NFκB-dependent kinase proviral integration site for Moloney murine leukemia virus-1 (PIM-1), which in turn regulates NFκB and Eomes. T cells defective in TCR-dependent NFκB signaling were impaired in late expression of Pim-1, Eomes, and CD8 memory. These defects were rescued when TCR-dependent NFκB signaling was restored. We also found that NFκB-Pim-1 signals were required at memory to maintain memory CD8 T-cell longevity, effector function, and Eomes expression. Hence, an NFκB-Pim-1-Eomes axis regulates Eomes levels to maintain memory fitness.

Studying assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy.

Significant progress has been made in obtaining structural insight into the assembly of the ß-barrel assembly machinery complex (BAM). These crystallography and electron microscopy studies used detergent as a membrane mimetic and revealed structural variations in the central domain, BamA, as well as in the lipoprotein BamC. We have used cellular solid-state NMR spectroscopy to examine the entire BamABCDE complex in native outer membranes and obtained data on the BamCDE subcomplex in outer membranes, in addition to synthetic bilayers. To reduce spectral crowding, we utilized proton-detected experiments and employed amino-acid specific isotope-labelling in (13C, 13C) correlation experiments. Taken together, the results provide insight into the overall fold and assembly of the BAM complex in native membranes, in particular regarding the structural flexibility of BamC in the absence of the core unit BamA.

Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR.

Studying biomolecules at atomic resolution in their native environment is the ultimate aim of structural biology. We investigated the bacterial type IV secretion system core complex (T4SScc) by cellular dynamic nuclear polarization-based solid-state nuclear magnetic resonance spectroscopy to validate a structural model previously generated by combining in vitro and in silico data. Our results indicate that T4SScc is well folded in the cellular setting, revealing protein regions that had been elusive when studied in vitro.

Aptamer-displaying peptide amphiphile micelles as a cell-targeted delivery vehicle of peptide cargoes.

Peptide amphiphile micelles (PAMs) are attractive vehicles for the delivery of a variety of therapeutic and prophylactic peptides. However, a key limitation of PAMs is their lack of preferential targeting ability. In this paper, we describe our design of a PAM system that incorporates a DNA oligonucleotide amphiphile (antitail amphiphile-AA) to form A/PAMs. A cell-targeting DNA aptamer with a 3' extension sequence (tail) complementary to the AA is annealed to the surface to form aptamer-displaying PAMs (Aptamer~A/PAMs). Aptamer~A/PAMs are small, anionic, stable nanoparticles capable of delivering a large mass percentage peptide amphiphile (PA) compared to targeting DNA components. Aptamer~A/PAMs are stable for over 4 h in the presence of biological fluids. Additionally, the aptamer retains its cell-targeting properties when annealed to the A/PAM, thus leading to enhanced delivery to a specifically-targeted B-cell leukemia cell line. This exciting modular technology can be readily used with a library of different targeting aptamers and PAs, capable of improving the bioavailability and potency of the peptide cargo.

POSH Regulates CD4+ T Cell Differentiation and Survival.

The scaffold molecule POSH is crucial for the regulation of proliferation and effector function in CD8(+) T cells. However, its role in CD4(+) T cells is not known. In this study, we found that disruption of the POSH scaffold complex established a transcriptional profile that strongly skewed differentiation toward Th2, led to decreased survival, and had no effect on cell cycle entry. This is in stark contrast to CD8(+) T cells in which POSH regulates cell cycle and does not affect survival. Disruption of POSH in CD4(+) T cells resulted in the loss of Tak1-dependent activation of JNK1/2 and Tak1-mediated survival. However, in CD8(+) T cells, POSH regulates only JNK1. Remarkably, each type of T cell had a unique composition of the POSH scaffold complex and distinct posttranslational modifications of POSH. These data indicate that the mechanism that regulates POSH function in CD4(+) T cells is different from CD8(+) T cells. All together, these data strongly suggest that POSH is essential for the integration of cell-type-specific signals that regulate the differentiation, survival, and function of T cells.

IL-12 Signals through the TCR To Support CD8 Innate Immune Responses.

CD8 T cells must integrate antigenic and inflammatory signals to differentiate into efficient effector and memory T cells able to protect us from infections. The mechanisms by which TCR signaling and proinflammatory cytokine receptor signaling cooperate in these processes are poorly defined. In this study, we show that IL-12 and other proinflammatory cytokines transduce signals through the TCR signalosome in a manner that requires Fyn activity and self-peptide-MHC (self-pMHC) interactions. This mechanism is crucial for CD8 innate T cell functions. Loss of Fyn activity or blockade of self-pMHC interactions severely impaired CD8 T cell IFN-γ and NKG2D expression, proliferation, and cytotoxicity upon cytokine-mediated bystander activation. Most importantly, in the absence of self-pMHC interactions, CD8 memory T cells fail to undergo bystander activation upon an unrelated infection. Thus, CD8 T cell bystander activation, although independent of cognate Ag, still requires self-pMHC and TCR signaling.

A Multicenter, Prospective, Randomized, Contralateral Study of Tissue Liquefaction Liposuction vs Suction-Assisted Liposuction.

BACKGROUND: Tissue liquefaction liposuction (TLL) deploys a novel energy source utilizing a stream of warmed, low-pressurized, and pulsed saline to extract fat tissue. OBJECTIVES: Compare TLL to suction-assisted liposuction (SAL) to determine which device is more efficient for surgeons and provides better recovery for patients. METHODS: Thirty-one adult female patients were followed prospectively in a contralateral study design comparing differences in bruising, swelling, tenderness, and incision appearance ratings between TLL and SAL procedures. Surgical efficiency and appearance of the lipoaspirate were also compared. RESULTS: All 31 patients successfully completed the study. For TLL and SAL procedures, the average volumes of infusion (1.242 vs 1.276 L) and aspirated supernatant fat (704 vs 649 mL) were statistically similar. TLL median fat extraction rate was faster than SAL (35.6 vs 25 mL/min; P < 0.0001), and stroke rate was reduced in TLL vs SAL procedures (48 vs 120 strokes/min; P < 0.0001), and both were statistically significant. The mean total scores for bruising, swelling, treatment site tenderness, and incision appearance were lower, indicating improved patient recovery on the TLL side. CONCLUSIONS: TLL and SAL techniques produced comparable volume of fat aspirate. TLL demonstrated a 42% faster fat extraction rate and a 68% reduction in arm movements needed to complete the procedure compared to SAL, both of these differences are statistically significant. The TLL side was noted to have reduced bruising and swelling and improved incision site appearance with less tenderness compared to the SAL side.

Supramolecular Organization and Functional Implications of K+ †Channel Clusters in Membranes.

The segregation of cellular surfaces in heterogeneous patches is considered to be a common motif in bacteria and eukaryotes that is underpinned by the observation of clustering and cooperative gating of signaling membrane proteins such as receptors or channels. Such processes could represent an important cellular strategy to shape signaling activity. Hence, structural knowledge of the arrangement of channels or receptors in supramolecular assemblies represents a crucial step towards a better understanding of signaling across membranes. We herein report on the supramolecular organization of clusters of the K+ channel KcsA in bacterial membranes, which was analyzed by a combination of DNP-enhanced solid-state NMR experiments and MD simulations. We used solid-state NMR spectroscopy to determine the channel-channel interface and to demonstrate the strong correlation between channel function and clustering, which suggests a yet unknown mechanism of communication between K+ channels.

Structural basis of nucleic acid binding by Nicotiana tabacum glycine-rich RNA-binding protein: implications for its RNA chaperone function.

Glycine-rich RNA-binding proteins (GR-RBPs) are involved in cold shock response of plants as RNA chaperones facilitating mRNA transport, splicing and translation. GR-RBPs are bipartite proteins containing a RNA recognition motif (RRM) followed by a glycine-rich region. Here, we studied the structural basis of nucleic acid binding of full-length Nicotiana tabacum GR-RBP1. NMR studies of NtGR-RBP1 show that the glycine-rich domain, while intrinsically disordered, is responsible for mediating self-association by transient interactions with its RRM domain (NtRRM). Both NtGR-RBP1 and NtRRM bind specifically and with low micromolar affinity to RNA and single-stranded DNA. The solution structure of NtRRM shows that it is a canonical RRM domain. A HADDOCK model of the NtRRM-RNA complex, based on NMR chemical shift and NOE data, shows that nucleic acid binding results from a combination of stacking and electrostatic interactions with conserved RRM residues. Finally, DNA melting experiments demonstrate that NtGR-RBP1 is more efficient in melting CTG containing nucleic acids than isolated NtRRM. Together, our study supports the model that self-association of GR-RBPs by the glycine-rich region results in cooperative unfolding of non-native substrate structures, thereby enhancing its chaperone function.

1 H-Detected Solid-State NMR Studies of Water-Inaccessible Proteins In†Vitro and In†Situ.

1 H detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for 1 H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water-inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high-resolution 1 H-detected solid-state NMR (ssNMR) studies of water-inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K+ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K+ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.

Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

Cutting edge: The signals for the generation of T cell memory are qualitatively different depending on TCR ligand strength.

CD8 T cell memory critically contributes to long-term immunity. Both low- and high-affinity TCR signals are able to support the differentiation of memory CD8 T cells. However, it is unclear whether the requirements for memory development change when TCR signal strength is altered. To gain further insight into this question, we used a TCRß transmembrane domain mutant model that is defective in the generation of memory in response to high-affinity ligands. Surprisingly, lowering TCR signal strength, by stimulation with low-affinity ligands, resulted in normal memory development. Restoration of memory correlated with recovery of TCR-dependent NF-κB signaling. Thus, these data provide novel evidence that the requirements for memory are qualitatively different depending on TCR signal strength.