US oil and gas system emissions from nearly one million aerial site measurements.

As airborne methane surveys of oil and gas systems continue to discover large emissions that are missing from official estimates1-4, the true scope of methane emissions from energy production has yet to be quantified. We integrate approximately one million aerial site measurements into regional emissions inventories for six regions in the USA, comprising 52% of onshore oil and 29% of gas production over 15 aerial campaigns. We construct complete emissions distributions for each, employing empirically grounded simulations to estimate small emissions. Total estimated emissions range from 0.75% (95% confidence interval (CI) 0.65%, 0.84%) of covered natural gas production in a high-productivity, gas-rich region to 9.63% (95% CI 9.04%, 10.39%) in a rapidly expanding, oil-focused region. The six-region weighted average is 2.95% (95% CI 2.79%, 3.14%), or roughly three times the national government inventory estimate5. Only 0.05-1.66% of well sites contribute the majority (50-79%) of well site emissions in 11 out of 15 surveys. Ancillary midstream facilities, including pipelines, contribute 18-57% of estimated regional emissions, similarly concentrated in a small number of point sources. Together, the emissions quantified here represent an annual loss of roughly US$1 billion in commercial gas value and a US$9.3 billion annual social cost6. Repeated, comprehensive, regional remote-sensing surveys offer a path to detect these low-frequency, high-consequence emissions for rapid mitigation, incorporation into official emissions inventories and a clear-eyed assessment of the most effective emission-finding technologies for a given region.

Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

ADAR1 masks the cancer immunotherapeutic promise of ZBP1-driven necroptosis.

Only a small proportion of patients with cancer show lasting responses to immune checkpoint blockade (ICB)-based monotherapies. The RNA-editing enzyme ADAR1 is an emerging determinant of resistance to ICB therapy and prevents ICB responsiveness by repressing immunogenic double-stranded RNAs (dsRNAs), such as those arising from the dysregulated expression of endogenous retroviral elements (EREs)1-4. These dsRNAs trigger an interferon-dependent antitumour response by activating A-form dsRNA (A-RNA)-sensing proteins such as MDA-5 and PKR5. Here we show that ADAR1 also prevents the accrual of endogenous Z-form dsRNA elements (Z-RNAs), which were enriched in the 3' untranslated regions of interferon-stimulated mRNAs. Depletion or mutation of ADAR1 resulted in Z-RNA accumulation and activation of the Z-RNA sensor ZBP1, which culminated in RIPK3-mediated necroptosis. As no clinically viable ADAR1 inhibitors currently exist, we searched for a compound that can override the requirement for ADAR1 inhibition and directly activate ZBP1. We identified a small molecule, the curaxin CBL0137, which potently activates ZBP1 by triggering Z-DNA formation in cells. CBL0137 induced ZBP1-dependent necroptosis in cancer-associated fibroblasts and reversed ICB unresponsiveness in mouse models of melanoma. Collectively, these results demonstrate that ADAR1 represses endogenous Z-RNAs and identifies ZBP1-mediated necroptosis as a new determinant of tumour immunogenicity masked by ADAR1. Therapeutic activation of ZBP1-induced necroptosis provides a readily translatable avenue for rekindling the immune responsiveness of ICB-resistant human cancers.

The intracellular domain of Sema6A is essential for development of the zebrafish retina.

Semaphorin6A (Sema6A) is a repulsive guidance molecule that plays many roles in central nervous system, heart and bone development, as well as immune system responses and cell signaling in cancer. Loss of Sema6A or its receptor PlexinA2 in zebrafish leads to smaller eyes and improper retinal patterning. Here, we investigate a potential role for the Sema6A intracellular domain in zebrafish eye development and dissect which phenotypes rely on forward signaling and which rely on reverse signaling. We performed rescue experiments on zebrafish Sema6A morphants with either full-length Sema6A (Sema6A-FL) or Sema6A lacking its intracellular domain (Sema6A-ΔC). We identified that the intracellular domain is not required for eye size and retinal patterning, however it is required for retinal integrity, the number and end feet strength of Müller glia and protecting against retinal cell death. This novel function for the intracellular domain suggests a role for Sema6A reverse signaling in zebrafish eye development.

California's methane super-emitters.

Methane is a powerful greenhouse gas and is targeted for emissions mitigation by the US state of California and other jurisdictions worldwide1,2. Unique opportunities for mitigation are presented by point-source emitters-surface features or infrastructure components that are typically less than 10 metres in diameter and emit plumes of highly concentrated methane3. However, data on point-source emissions are sparse and typically lack sufficient spatial and temporal resolution to guide their mitigation and to accurately assess their magnitude4. Here we survey more than 272,000 infrastructure elements in California using an airborne imaging spectrometer that can rapidly map methane plumes5-7. We conduct five campaigns over several months from 2016 to 2018, spanning the oil and gas, manure-management and waste-management sectors, resulting in the detection, geolocation and quantification of emissions from 564 strong methane point sources. Our remote sensing approach enables the rapid and repeated assessment of large areas at high spatial resolution for a poorly characterized population of methane emitters that often appear intermittently and stochastically. We estimate net methane point-source emissions in California to be 0.618 teragrams per year (95 per cent confidence interval 0.523-0.725), equivalent to 34-46 per cent of the state's methane inventory8 for 2016. Methane 'super-emitter' activity occurs in every sector surveyed, with 10 per cent of point sources contributing roughly 60 per cent of point-source emissions-consistent with a study of the US Four Corners region that had a different sectoral mix9. The largest methane emitters in California are a subset of landfills, which exhibit persistent anomalous activity. Methane point-source emissions in California are dominated by landfills (41 per cent), followed by dairies (26 per cent) and the oil and gas sector (26 per cent). Our data have enabled the identification of the 0.2 per cent of California's infrastructure that is responsible for these emissions. Sharing these data with collaborating infrastructure operators has led to the mitigation of anomalous methane-emission activity10.

Strong methane point sources contribute a disproportionate fraction of total emissions across multiple basins in the United States.

Understanding, prioritizing, and mitigating methane (CH4) emissions requires quantifying CH4 budgets from facility scales to regional scales with the ability to differentiate between source sectors. We deployed a tiered observing system for multiple basins in the United States (San Joaquin Valley, Uinta, Denver-Julesburg, Permian, Marcellus). We quantify strong point source emissions (>10 kg CH4 h-1) using airborne imaging spectrometers, attribute them to sectors, and assess their intermittency with multiple revisits. We compare these point source emissions to total basin CH4 fluxes derived from inversion of Sentinel-5p satellite CH4 observations. Across basins, point sources make up on average 40% of the regional flux. We sampled some basins several times across multiple months and years and find a distinct bimodal structure to emission timescales: the total point source budget is split nearly in half by short-lasting and long-lasting emission events. With the increasing airborne and satellite observing capabilities planned for the near future, tiered observing systems will more fully quantify and attribute CH4 emissions from facility to regional scales, which is needed to effectively and efficiently reduce methane emissions.

Vδ1 Effector and Vδ2 γδ T-Cell Subsets Shift in Frequency and Are Linked to Plasma Inflammatory Markers During Antiretroviral Therapy-Suppressed HIV Infection.

BACKGROUND: Chronic inflammation is prevalent with antiretroviral therapy (ART)-suppressed human immunodeficiency virus (HIV) infection and one immune cell subset putatively driving this phenomenon is TIGIT+ γδ T cells. METHODS: To elucidate γδ T-cell phenotypic diversity, spectral flow cytometry was performed on blood lymphocytes from individuals of a HIV and aging cohort and data were analyzed using bioinformatic platforms. Plasma inflammatory markers were measured and correlated with γδ T-cell subset frequencies. RESULTS: Thirty-nine distinct γδ T-cell subsets were identified (22 Vδ1+, 14 Vδ2+, and 3 Vδ1-Vδ2-Vγ9+) and TIGIT was nearly exclusively found on the Vδ1+CD45RA+CD27- effector populations. People with ART-suppressed HIV infection (PWH) exhibited high frequencies of distinct clusters of Vδ1+ effectors distinguished via CD8, CD16, and CD38 expression. Among Vδ2+ cells, most Vγ9+ (innate-like) clusters were lower in PWH; however, CD27+ subsets were similar in frequency between participants with and without HIV. Comparisons by age revealed lower 'naive' Vδ1+CD45RA+CD27+ cells in older individuals, regardless of HIV status. Plasma inflammatory markers were selectively linked to subsets of Vδ1+ and Vδ2+ cells. CONCLUSIONS: These results further elucidate γδ T-cell subset complexity and reveal distinct alterations and connections with inflammatory pathways of Vδ1+ effector and Vδ2+ innate-like subsets during ART-suppressed HIV infection.

A General Synthetic Strategy toward the Truxillate Natural Products via Solid-State Photocycloadditions.

The truxillates constitute a large class of dimeric natural products featuring a central, highly substituted cyclobutane core. In principle, these structures could be efficiently synthesized via [2 + 2] photocycloaddition. However, the difficulty in controlling the high-energy electronically excited reactive intermediates in the solution state can lead to poor regio- and diastereocontrol. This has limited the use of photocycloaddition methodology toward the synthesis of this important class of natural products. Herein, we demonstrate that acid-controlled precipitation of C-acyl imidazoles promotes a highly selective solid-state photocycloaddition, and the products of this reaction can be quickly transformed into truxillate natural products.

Small molecule inhibitors of transcriptional cyclin-dependent kinases impose HIV-1 latency, presenting "block and lock" treatment strategies.

Current antiretroviral therapy for HIV-1 infection does not represent a cure for infection as viral rebound inevitably occurs following discontinuation of treatment. The "block and lock" therapeutic strategy is intended to enforce proviral latency and durably suppress viremic reemergence in the absence of other intervention. The transcription-associated cyclin-dependent protein kinases (tCDKs) are required for expression from the 5´ HIV-1 long-terminal repeat, but the therapeutic potential of inhibiting these kinases for enforcing HIV-1 latency has not been characterized. Here, we expanded previous observations to directly compare the effect of highly selective small molecule inhibitors of CDK7 (YKL-5-124), CDK9 (LDC000067), and CDK8/19 (Senexin A), and found each of these prevented HIV-1 provirus expression at concentrations that did not cause cell toxicity. Inhibition of CDK7 caused cell cycle arrest, whereas CDK9 and CDK8/19 inhibitors did not, and could be continuously administered to establish proviral latency. Upon discontinuation of drug administration, HIV immediately rebounded in cells that had been treated with the CDK9 inhibitor, while proviral latency persisted for several days in cells that had been treated with CDK8/19 inhibitors. These results identify the mediator kinases CDK8/CDK19 as potential "block and lock" targets for therapeutic suppression of HIV-1 provirus expression.

Conceptus Estrogen and prostaglandins provide the maternal recognition of pregnancy signal to prevent Luteolysis during early pregnancy in the pig†.

Conceptus estrogens and prostaglandins have long been considered the primary signals for maternal recognition of pregnancy (MRP) in the pig. However, loss-of-function studies targeting conceptus aromatase genes (CYP19A1 and CYP19A2) and prostaglandin-endoperoxide synthase 2 (PTGS2) indicated that conceptuses can not only signal MRP without estrogens or prostaglandins but can maintain early pregnancy. However, complete loss of estrogen production leads to abortion after day 25 of gestation. Although neither conceptus estrogens nor prostaglandins had a significant effect on early maintenance of CL function alone, the two conceptus factors have a biological relationship. To investigate the role that both conceptus estrogens and prostaglandins have on MRP and maintenance of pregnancy, a triple loss-of function model (TKO) was generated for conceptus CYP19A1, CYP19A2 and PTGS2. In addition, a conceptus CYP19A2-/- model (A2KO) was established to determine the role of placental estrogen during later pregnancy. Estrogen and prostaglandin synthesis were greatly reduced in TKO conceptuses which resulted in a failure to inhibit luteolysis after day 15 of pregnancy despite the presence of conceptuses in the uterine lumen. However, A2KO placentae not only maintained functional CL but were able to maintain pregnancy to day 32 of gestation. Despite the loss of placental CYP19A2 expression, the allantois fluid content of estrogen was not affected as the placenta compensated by expressing CYP19A1 and CYP19A3, which are normally absent in controls. Results suggest conceptuses can signal MRP through production of conceptus PGE or stimulating PGE synthesis from the endometrium through conceptus estrogen. Failure of conceptuses to produce both factors results in failure of MRP and loss of pregnancy.

CDK8 inhibitors antagonize HIV-1 reactivation and promote provirus latency in T cells.

Latent HIV-1 provirus represents the barrier toward a cure for infection and is dependent upon the host RNA Polymerase (Pol) II machinery for reemergence. Here, we find that inhibitors of the RNA Pol II mediator kinases CDK8/19, Senexin A and BRD6989, inhibit induction of HIV-1 expression in response to latency-reversing agents and T cell signaling agonists. These inhibitors were found to impair recruitment of RNA Pol II to the HIV-1 LTR. Furthermore, HIV-1 expression in response to several latency reversal agents was impaired upon disruption of CDK8 by shRNA or gene knockout. However, the effects of CDK8 depletion did not entirely mimic CDK8/19 kinase inhibition suggesting that the mediator kinases are not functionally redundant. Additionally, treatment of CD4+ peripheral blood mononuclear cells isolated from people living with HIV-1 and who are receiving antiretroviral therapy with Senexin A inhibited induction of viral replication in response to T cell stimulation by PMA and ionomycin. These observations indicate that the mediator kinases, CDK8 and CDK19, play a significant role for regulation of HIV-1 transcription and that small molecule inhibitors of these enzymes may contribute to therapies designed to promote deep latency involving the durable suppression of provirus expression. IMPORTANCE A cure for HIV-1 infection will require novel therapies that can force elimination of cells that contain copies of the virus genome inserted into the cell chromosome, but which is shut off, or silenced. These are known as latently-infected cells, which represent the main reason why current treatment for HIV/AIDS cannot cure the infection because the virus in these cells is unaffected by current drugs. Our results indicate that chemical inhibitors of Cdk8 also inhibit the expression of latent HIV provirus. Cdk8 is an important enzyme that regulates the expression of genes in response to signals to which cells need to respond and which is produced by a gene that is frequently mutated in cancers. Our observations indicate that Cdk8 inhibitors may be employed in novel therapies to prevent expression from latent provirus, which might eventually enable infected individuals to cease treatment with antiretroviral drugs.

Augmentation of tumor expression of HLA-DR, CXCL9, and CXCL10 may improve olfactory neuroblastoma immunotherapeutic responses.

BACKGROUND: Olfactory neuroblastoma is a rare malignancy of the anterior skull base typically treated with surgery and adjuvant radiation. Although outcomes are fair for low-grade disease, patients with high-grade, recurrent, or metastatic disease oftentimes respond poorly to standard treatment methods. We hypothesized that an in-depth evaluation of the olfactory neuroblastoma tumor immune microenvironment would identify mechanisms of immune evasion in high-grade olfactory neuroblastoma as well as rational targetable mechanisms for future translational immunotherapeutic approaches. METHODS: Multispectral immunofluorescence and RNAScope evaluation of the tumor immune microenvironment was performed on forty-seven clinically annotated olfactory neuroblastoma samples. A retrospective chart review was performed and clinical correlations assessed. RESULTS: A significant T cell infiltration was noted in olfactory neuroblastoma samples with a stromal predilection, presence of myeloid-derived suppressor cells, and sparse natural killer cells. A striking decrease was observed in MHC-I expression in high-grade olfactory neuroblastoma compared to low-grade disease, representing a mechanism of immune evasion in high-grade disease. Mechanistically, the immune effector stromal predilection appears driven by low tumor cell MHC class II (HLA-DR), CXCL9, and CXCL10 expression as those tumors with increased tumor cell expression of each of these mediators correlated with significant increases in T cell infiltration. CONCLUSION: These data suggest that immunotherapeutic strategies that augment tumor cell expression of MHC class II, CXCL9, and CXCL10 may improve parenchymal trafficking of immune effector cells in olfactory neuroblastoma and augment immunotherapeutic responses.

Quantitative image analysis pipeline for detecting circulating hybrid cells in immunofluorescence images with human-level accuracy.

Circulating hybrid cells (CHCs) are a newly discovered, tumor-derived cell population found in the peripheral blood of cancer patients and are thought to contribute to tumor metastasis. However, identifying CHCs by immunofluorescence (IF) imaging of patient peripheral blood mononuclear cells (PBMCs) is a time-consuming and subjective process that currently relies on manual annotation by laboratory technicians. Additionally, while IF is relatively easy to apply to tissue sections, its application to PBMC smears presents challenges due to the presence of biological and technical artifacts. To address these challenges, we present a robust image analysis pipeline to automate the detection and analysis of CHCs in IF images. The pipeline incorporates quality control to optimize specimen preparation protocols and remove unwanted artifacts, leverages a ß-variational autoencoder (VAE) to learn meaningful latent representations of single-cell images, and employs a support vector machine (SVM) classifier to achieve human-level CHC detection. We created a rigorously labeled IF CHC data set including nine patients and two disease sites with the assistance of 10 annotators to evaluate the pipeline. We examined annotator variation and bias in CHC detection and provided guidelines to optimize the accuracy of CHC annotation. We found that all annotators agreed on CHC identification for only 65% of the cells in the data set and had a tendency to underestimate CHC counts for regions of interest (ROIs) containing relatively large amounts of cells (>50,000) when using the conventional enumeration method. On the other hand, our proposed approach is unbiased to ROI size. The SVM classifier trained on the ß-VAE embeddings achieved an F1 score of 0.80, matching the average performance of human annotators. Our pipeline enables researchers to explore the role of CHCs in cancer progression and assess their potential as a clinical biomarker for metastasis. Further, we demonstrate that the pipeline can identify discrete cellular phenotypes among PBMCs, highlighting its utility beyond CHCs.

Reducing ambulatory central line-associated bloodstream infections: A family-centered approach.

BACKGROUND: Ambulatory central line-associated bloodstream infections (CLABSIs) cause significant morbidity and mortality, especially in pediatric oncology. Few studies have had interventions directed toward caregivers managing central lines (CL) at home to reduce ambulatory CLABSI rates. We aimed to reduce and sustain our ambulatory CLABSI rate by 25% within 3 years of the start of a quality improvement intervention. PROCEDURE: Plan-do-study-act cycles were implemented beginning April 2016. The main intervention was a family-centered CL care skill development curriculum for external CLs. Training began upon hospital CL insertion, followed by an ambulatory teach-back program to achieve home caregiver CL care independence. Other changes included: standardizing ambulatory nurse CL care practice (audits, a train the nurse trainer process, and workshops for independent home care agencies); developing aids for trainers and caregivers; providing supplies for clean surfaces; wide dissemination of the program; and minimizing opportunities of CLABSI (e.g., standardizing timing of CL removal). The outcome measure was the ambulatory CLABSI rate (excluding mucosal barrier injury laboratory-confirmed bloodstream infection), compared pre intervention (January 2015 to March 2016) to post intervention, including 2 years of sustainability (April 2016 to June 2023), using statistical process control charts. We estimated the total number of CLABSI and associated healthcare charges prevented. RESULTS: The ambulatory CLABSI rate decreased by 52% from 0.25 to 0.12 per 1000 CL days post intervention, achieved within 27 months; 117 CLABSI were prevented, with $4.2 million hospital charges and 702 hospital days avoided. CONCLUSIONS: Focusing efforts on home caregivers CL care may lead to reduction in pediatric oncology ambulatory CLABSI rates.

A Comparative Assessment of Outcomes Following Mandibular Distraction Osteogenesis and Tongue-Lip Adhesion in Pierre Robin Sequence.

The purpose of this study was to assess long-term outcomes of tongue-lip adhesion (TLA) and mandibular distraction osteogenesis (MDO) to resolve upper airway obstruction in patients with Robin sequence (RS). A retrospective cohort study was performed of subjects presenting to a tertiary care pediatric center who underwent either primary MDO or TLA for the treatment of RS between 2004 and 2020. N=59 subjects met inclusion criteria (n=34 MDO, n=25 TLA), and there were no significant differences in preoperative patient characteristics other than age at surgery (MDO 31 d vs. TLA 17 d, P=0.049). Preoperative apnea-hypopnea index (AHI) was similar between cohorts (33.9 and 46.7, P=0.38). Subjects who underwent MDO demonstrated improved AHI on initial postoperative polysomnogram performed at 2 weeks (3.4 vs. 11.6, P=0.014), however AHI at the second postoperative timepoint (270 vs. 142 d, P=0.007) was no different between cohorts (2.8 vs. 2.6, P=0.89). No subject in either group required enteral nutrition or supplemental oxygen at last follow-up. In subjects undergoing MDO, 14.7% demonstrated temporary asymmetric marginal mandibular nerve dysfunction. Forty-seven percent of MDO patients had injury to first primary molars. MDO and TLA both ultimately achieved similar long-term resolution of upper airway obstruction and associated feeding difficulties in patients with Robin sequence. MDO offered a more immediate airway improvement, but the procedure carried a potential risk of neurosensory and dental injury when compared with TLA.

Farnesol remodels the peritoneal cavity immune environment influencing Candida albicans pathogenesis during intra-abdominal infection.

Candida albicans is a lifelong member of the mycobiome causing mucosal candidiasis and life-threatening, systemic, and intra-abdominal disease in immunocompromised and transplant patients. Despite the clinical importance of intra-abdominal candidiasis with mortality rates between 40% and 70%, the contribution of fungal virulence factors and host immune responses to disease has not been extensively studied. Secretion of the quorum-sensing molecule, farnesol, acts as a virulence factor for C. albicans during systemic infection, while inducing local, protective innate immune responses in oral models of infection. Previously, we reported that farnesol recruits macrophages to the peritoneal cavity in mice, suggesting a role for farnesol in innate immune responses. Here, we expand on our initial findings, showing that farnesol profoundly alters the peritoneal cavity microenvironment promoting innate inflammation. Intra-peritoneal injection of farnesol stimulates rapid local death of resident peritoneal cells followed by recruitment of neutrophils and inflammatory macrophages into the peritoneal cavity and peritoneal mesothelium associated with an early increase in chemokines followed by proinflammatory cytokines. These rapid inflammatory responses to farnesol significantly increase morbidity and mortality of mice with intra-abdominal candidiasis associated with increased formation of peritoneal adhesions, despite similar rates of fungal clearance from the peritoneal cavity and retro-peritoneal organs. C. albicans ddp3Δ/ddp3Δ knockout and reconstituted strains recapitulate these findings. This indicates that farnesol may be detrimental to the host during intra-abdominal infections. Importantly, our results highlight a need to understand how C. albicans virulence factors modulate the host immune response within the peritoneum, an exceedingly common site of Candida infection.

Pathogenic Variants in the Myosin Chaperone UNC-45B Cause Progressive Myopathy with Eccentric Cores.

The myosin-directed chaperone UNC-45B is essential for sarcomeric organization and muscle function from Caenorhabditis elegans to humans. The pathological impact of UNC-45B in muscle disease remained elusive. We report ten individuals with bi-allelic variants in UNC45B who exhibit childhood-onset progressive muscle weakness. We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization.

Truncation-Free Genetic Code Expansion with Tetrazine Amino Acids for Quantitative Protein Ligations.

Quantitative labeling of biomolecules is necessary to advance areas of antibody-drug conjugation, super-resolution microscopy imaging of molecules in live cells, and determination of the stoichiometry of protein complexes. Bio-orthogonal labeling to genetically encodable noncanonical amino acids (ncAAs) offers an elegant solution; however, their suboptimal reactivity and stability hinder the utility of this method. Previously, we showed that encoding stable 1,2,4,5-tetrazine (Tet)-containing ncAAs enables rapid, complete conjugation, yet some expression conditions greatly limited the quantitative reactivity of the Tet-protein. Here, we demonstrate that reduction of on-protein Tet ncAAs impacts their reactivity, while the leading cause of the unreactive protein is near-cognate suppression (NCS) of UAG codons by endogenous aminoacylated tRNAs. To overcome incomplete conjugation due to NCS, we developed a more catalytically efficient tRNA synthetase and developed a series of new machinery plasmids harboring the aminoacyl tRNA synthetase/tRNA pair (aaRS/tRNA pair). These plasmids enable robust production of homogeneously reactive Tet-protein in truncation-free cell lines, eliminating the contamination caused by NCS and protein truncation. Furthermore, these plasmid systems utilize orthogonal synthetic origins, which render these machinery vectors compatible with any common expression system. Through developing these new machinery plasmids, we established that the aaRS/tRNA pair plasmid copy-number greatly affects the yields and quality of the protein produced. We then produced quantitatively reactive soluble Tet-Fabs, demonstrating the utility of this system for rapid, homogeneous conjugations of biomedically relevant proteins.

Spinal Cord Atrophy Predicts Progressive Disease in Relapsing Multiple Sclerosis.

OBJECTIVE: A major challenge in multiple sclerosis (MS) research is the understanding of silent progression and Progressive MS. Using a novel method to accurately capture upper cervical cord area from legacy brain MRI scans we aimed to study the role of spinal cord and brain atrophy for silent progression and conversion to secondary progressive disease (SPMS). METHODS: From a single-center observational study, all RRMS (n = 360) and SPMS (n = 47) patients and 80 matched controls were evaluated. RRMS patient subsets who converted to SPMS (n = 54) or silently progressed (n = 159), respectively, during the 12-year observation period were compared to clinically matched RRMS patients remaining RRMS (n = 54) or stable (n = 147), respectively. From brain MRI, we assessed the value of brain and spinal cord measures to predict silent progression and SPMS conversion. RESULTS: Patients who developed SPMS showed faster cord atrophy rates (-2.19%/yr) at least 4 years before conversion compared to their RRMS matches (-0.88%/yr, p < 0.001). Spinal cord atrophy rates decelerated after conversion (-1.63%/yr, p = 0.010) towards those of SPMS patients from study entry (-1.04%). Each 1% faster spinal cord atrophy rate was associated with 69% (p < 0.0001) and 53% (p < 0.0001) shorter time to silent progression and SPMS conversion, respectively. INTERPRETATION: Silent progression and conversion to secondary progressive disease are predominantly related to cervical cord atrophy. This atrophy is often present from the earliest disease stages and predicts the speed of silent progression and conversion to Progressive MS. Diagnosis of SPMS is rather a late recognition of this neurodegenerative process than a distinct disease phase. ANN NEUROL 2022;91:268-281.

Examining Between-Group Differences in Social Network Density and High-Sensitivity C-Reactive Protein in Older Adults: Implications for the Hispanic Mortality Paradox.

OBJECTIVE: Hispanic/Latino(x) and African American/Black older adults experience disproportionate cardiometabolic disease burdens when compared with their non-Hispanic White counterparts. Sources of resilience such as social networks have been found to mitigate the risk of this disease and its end points like high-sensitivity C-reactive protein (hs-CRP). However, little is known about the social network infrastructure among these groups. Moreover, existing work has largely ignored the degree to which members of one's network are connected to one another (network density), which may be important for navigating structural barriers within interdependent groups. The objective of this study was to understand the association between network density and 5-year hs-CRP (blood spot) and whether this association was moderated by race-ethnicity. METHODS: A subsample of Hispanic/Latino(x), African American/Black, and non-Hispanic White older adults ( N = 1431) from the National Social Life Health and Aging Project was used. Multivariable regression was used to estimate the association between network density and its interaction with race-ethnicity, with hs-CRP 5 years later. RESULTS: Although no main effect of network density on 5-year hs-CRP was found, results revealed a significant network density by race-ethnicity interaction (Wald χ2 (2, 1242) = 3.31, p = .037). Simple slopes analyses revealed that Hispanic/Latino(x) older adults with high network density had significantly lower hs-CRP levels when compared with their same-ethnic counterparts with low network density ( b = -0.73, standard error = 0.31, 95% confidence interval = -1.33 to -0.13, p = .018). CONCLUSIONS: Results demonstrate population-level differences in social network structure and differential associations of this infrastructure with health. Implications for the Hispanic Mortality Paradox are discussed.