Targeting Bcl6 in the TREX1 D18N murine model ameliorates autoimmunity by modulating T-follicular helper cells and germinal center B cells.

The Three Prime Repair EXonuclease I (TREX1) is critical for degrading post-apoptosis DNA. Mice expressing catalytically inactive TREX1 (TREX1 D18N) develop lupus-like autoimmunity due to chronic sensing of undegraded TREX1 DNA substrates, production of the inflammatory cytokines, and the inappropriate activation of innate and adaptive immunity. This study aimed to investigate Thelper (Th) dysregulation in the TREX1 D18N model system as a potential mechanism for lupus-like autoimmunity. Comparison of immune cells in secondary lymphoid organs, spleen and peripheral lymph nodes (LNs) between TREX1 D18N mice and the TREX1 null mice revealed that the TREX1 D18N mice exhibit a Th1 bias. Additionally, the T-follicular helper cells (Tfh) and the germinal celter (GC) B cells were also elevated in the TREX1 D18N mice. Targeting Bcl6, a lineage-defining transcription factor for Tfh and GC B cells, with a commercially available Bcl6 inhibitor, FX1, attenuated Tfh, GC, and Th1 responses, and rescued TREX1 D18N mice from autoimmunity. The study presents Tfh and GC B-cell responses as potential targets in autoimmunity and that Bcl6 inhibitors may offer therapeutic approach in TREX1-associated or other lupus-like diseases.

T Cells Produce IFN-α in the TREX1 D18N Model of Lupus-like Autoimmunity.

Autoimmunity can result when cells fail to properly dispose of DNA. Mutations in the three-prime repair exonuclease 1 (TREX1) cause a spectrum of human autoimmune diseases resembling systemic lupus erythematosus. The cytosolic dsDNA sensor, cyclic GMP-AMP synthase (cGAS), and the stimulator of IFN genes (STING) are required for pathogenesis, but specific cells in which DNA sensing and subsequent type I IFN (IFN-I) production occur remain elusive. In this study, we demonstrate that TREX1 D18N catalytic deficiency causes dysregulated IFN-I signaling and autoimmunity in mice. Moreover, we show that bone marrow-derived cells drive this process. We identify both innate immune and, surprisingly, activated T cells as sources of pathological IFN-α production. These findings demonstrate that TREX1 enzymatic activity is crucial to prevent inappropriate DNA sensing and IFN-I production in immune cells, including normally low-level IFN-α-producing cells. These results expand our understanding of DNA sensing and innate immunity in T cells and may have relevance to the pathogenesis of human disease caused by TREX1 mutation.

Obstetric hospital preparedness for a pandemic: an obstetric critical care perspective in response to COVID-19.

The Coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has had a rapid and deadly onset, spreading quickly throughout the world. Pregnant patients have had high mortality rates, perinatal losses, and Intensive Care Unit (ICU) admissions from acute respiratory syndrome Coronavirus (SARS-CoV) and Middle East respiratory syndrome Coronavirus (MERS-CoV) in the past. Potentially, a surge of patients may require hospitalization and ICU care beyond the capacity of the health care system. This article is to provide institutional guidance on how to prepare an obstetric hospital service for a pandemic, mass casualty, or natural disaster by identifying a care model and resources for a large surge of critically ill pregnant patients over a short time. We recommend a series of protocols, education, and simulation training, with a structured and tiered approach to match the needs for the patients, for hospitals specialized in obstetrics.

Exonuclease TREX1 degrades double-stranded DNA to prevent spontaneous lupus-like inflammatory disease.

The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes a monogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme.

Solid-State Nanopore Analysis of Diverse DNA Base Modifications Using a Modular Enzymatic Labeling Process.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.

Racial/Ethnic Disparities in the Association Between Preeclampsia Risk Factors and Preeclampsia Among Women Residing in Hawaii.

Objective To assess differences in the rates of preeclampsia among a multiethnic population in Hawaii. Methods We performed a retrospective study on statewide inpatient data for delivery hospitalizations in Hawaii between January 1995 and December 2013. Multivariable logistic regression was used to assess the impact of maternal race/ethnicity on the rates of preeclampsia after adjusting for age, multiple gestation, multiparity, chronic hypertension, pregestational diabetes, obesity and smoking. Results A total of 271,569 hospital discharges for delivery were studied. The rates of preeclampsia ranged from 2.0 % for Chinese to 4.6 % for Filipinos. Preeclampsia rates were higher among Native Hawaiians who are age <35 and non-obese (OR 1.54; 95 % CI 1.43-1.66), age ≥35 and non-obese (OR 2.31; 95 % CI 2.00-2.68), age ≥35 and obese (OR 1.80; 95 % CI 1.24-2.60); other Pacific Islanders who are age <35 and non-obese (OR 1.40; 95 % CI 1.27-1.54), age ≥35 and non-obese (OR 2.18; 95 % CI 1.79-2.64), age ≥35 and obese (OR 1.68; 95 % CI 1.14-2.49); and Filipinos who are age <35 and non-obese (OR 1.55; 95 % CI 1.43-1.67), age ≥35 and non-obese (OR 2.26; 95 % CI 1.97-2.60), age ≥35 and obese (OR 1.64; 95 % CI 1.04-2.59) compared to whites. Pregestational diabetes (OR 3.41; 95 % CI 3.02-3.85), chronic hypertension (OR 5.98; 95 % CI 4.98-7.18), and smoking (OR 1.19; 95 % CI 1.07-1.33) were also independently associated with preeclampsia. Conclusions for Practice In Hawaii, Native Hawaiians, other Pacific Islanders and Filipinos have a higher risk of preeclampsia compared to whites. For these high-risk ethnic groups, more frequent monitoring for preeclampsia may be needed.

Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 are associated with systemic lupus erythematosus.

TREX1 acts in concert with the SET complex in granzyme A-mediated apoptosis, and mutations in TREX1 cause Aicardi-Goutières syndrome and familial chilblain lupus. Here, we report monoallelic frameshift or missense mutations and one 3' UTR variant of TREX1 present in 9/417 individuals with systemic lupus erythematosus but absent in 1,712 controls (P = 4.1 x 10(-7)). We demonstrate that two mutant TREX1 alleles alter subcellular targeting. Our findings implicate TREX1 in the pathogenesis of SLE.

The TREX1 C-terminal region controls cellular localization through ubiquitination.

TREX1 is an autonomous 3'-exonuclease that degrades DNA to prevent inappropriate immune activation. The TREX1 protein is composed of 314 amino acids; the N-terminal 242 amino acids contain the catalytic domain, and the C-terminal region (CTR) localizes TREX1 to the cytosolic compartment. In this study, we show that TREX1 modification by ubiquitination is controlled by a highly conserved sequence in the CTR to affect cellular localization. Transfection of TREX1 deletion constructs into human cells demonstrated that this sequence is required for ubiquitination at multiple lysine residues through a "non-canonical" ubiquitin linkage. A proteomic approach identified ubiquilin 1 as a TREX1 CTR-interacting protein, and this interaction was verified in vitro and in vivo. Cotransfection studies indicated that ubiquilin 1 localizes TREX1 to cytosolic punctate structures dependent upon the TREX1 CTR and lysines within the TREX1 catalytic core. Several TREX1 mutants linked to the autoimmune diseases Aicardi-Goutières syndrome and systemic lupus erythematosus that exhibit full catalytic function were tested for altered ubiquitin modification and cellular localization. Our data show that these catalytically competent disease-causing TREX1 mutants exhibit differential levels of ubiquitination relative to WT TREX1, suggesting a novel mechanism of dysfunction. Furthermore, these differentially ubiquitinated disease-causing mutants also exhibit altered ubiquilin 1 co-localization. Thus, TREX1 post-translational modification indicates an additional mechanism by which mutations disrupt TREX1 biology, leading to human autoimmune disease.

Molecular fingerprinting of the podocyte reveals novel gene and protein regulatory networks.

A thorough characterization of the transcriptome and proteome of endogenous podocytes has been hampered by low cell yields during isolation. Here we describe a double fluorescent reporter mouse model combined with an optimized bead perfusion protocol and efficient single cell dissociation to yield more than 500,000 podocytes per mouse allowing for global, unbiased downstream applications. Combining mRNA and miRNA transcriptional profiling with quantitative proteomic analyses revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport, and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. Thus, our 'omics' approach will facilitate the discovery and integration of novel gene, protein, and organelle regulatory networks that deepen our systematic understanding of podocyte biology.

The TREX1 exonuclease R114H mutation in Aicardi-Goutières syndrome and lupus reveals dimeric structure requirements for DNA degradation activity.

Mutations in the TREX1 gene cause Aicardi-Goutières syndrome (AGS) and are linked to the autoimmune disease systemic lupus erythematosus. The TREX1 protein is a dimeric 3' DNA exonuclease that degrades DNA to prevent inappropriate immune activation. One of the most common TREX1 mutations, R114H, causes AGS as a homozygous and compound heterozygous mutation and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1 proteins containing R114H and the insertion mutations aspartate at position 201 (D201ins) and alanine at position 124 (A124ins), found in compound heterozygous AGS with R114H, were prepared and the DNA degradation activities were tested. The homodimer TREX1(R114H/R114H) exhibits a 23-fold reduced single-stranded DNA (ssDNA) exonuclease activity relative to TREX1(WT). The TREX1(D201ins/D201ins) and TREX1(A124ins/A124ins) exhibit more than 10,000-fold reduced ssDNA degradation activities. However, the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers exhibit activities 10-fold greater than the TREX1(R114H/R114H) homodimer during ssDNA and double-stranded DNA (dsDNA) degradation. These higher levels of activities measured in the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers are attributed to Arg-114 residues of TREX1(D201ins) and TREX1(A124ins) positioned at the dimer interface contributing to the active sites of the opposing TREX1(R114H) protomer. This interpretation is further supported by exonuclease activities measured for TREX1 enzymes containing R114A and R114K mutations. These biochemical data provide direct evidence for TREX1 residues in one protomer contributing to DNA degradation catalyzed in the opposing protomer and help to explain the dimeric TREX1 structure required for full catalytic competency.

A murine model of Denys-Drash syndrome reveals novel transcriptional targets of WT1 in podocytes.

The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations. To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli and identified excellent candidates that may modify podocyte differentiation and growth factor signaling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and Sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild-type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis.

Rapid separation of beryllium and lanthanide derivatives by capillary gas chromatography.

Previous studies describe derivatization of metal ions followed by analysis using gas chromatography, usually on packed columns. In many of these studies, stable and volatile derivatives were formed using fluorinated ß-diketonate reagents. This paper extends previous work by investigating separations of the derivatives on small-diameter capillary gas chromatography columns and exploring on-fiber, solid-phase microextraction derivatization techniques for beryllium. The ß-diketonate used for these studies was 1,1,1,2,2,6,6,7,7,7-decafluoro-3,5-heptanedione. Derivatization of lanthanides also required addition of a neutral donor, dibutyl sulfoxide, in addition to 1,1,1,2,2,6,6,7,7,7-decafluoro-3,5-heptanedione. Unoptimized separations on a 100-µm i.d. capillary column proved capable of rapid separations (within 15 min) of lanthanide derivatives that are adjacent to one another in the periodic table. Full-scan mass spectra were obtained from derivatives containing 5 ng of each lanthanide. Studies also developed a simple on-fiber solid-phase microextraction derivatization of beryllium. Beryllium could be analyzed in the presence of other alkali earth elements (Ba(II) and Sr(II)) without interference. Extension of the general approach was demonstrated for several additional elements (i.e. Cu(II), Cr(III), and Ga(III)).

Rapid separation of beryllium and lanthanide derivatives by capillary gas chromatography.

DOI: 10.1002/jssc.201200537 Europium(III) derivative formed by reaction of the europium ion with a fluorinated ß-diketonate reagent and a neutral donor. The resulting non-polar, thermally stable, and luminescent derivative partitions into cyclohexane upon formation and can be subjected to high-resolution capillary gas chromatography. This approach is used to separate derivatives of lanthanides that are adjacent to one another in the periodic table. In addition, an on-fiber SPME derivatization/concentration GC/MS method that uses a fiber preloaded with the same ß-diketonate reagent is described for the analysis of beryllium(II) ion.

Racial/Ethnic Disparities in Intensive Care Admissions in a Pregnant and Postpartum Population, Hawai'i, 2012-2017.

OBJECTIVE: Maternal morbidity and mortality is a global concern despite advances in medical care and technology and improved economic resources of nations worldwide. The primary objective of our study was to describe racial/ethnic disparities in severe maternal morbidity by using admission to an intensive care unit (ICU) as a marker. The secondary objective was to evaluate associations between patient characteristics, including obstetric outcomes, and severe maternal morbidity. METHODS: This retrospective cohort study used a large inpatient database to identify pregnancy and postpartum hospitalizations in Hawai'i from January 2012 through September 2017. We evaluated associations between sociodemographic and clinical characteristics and race/ethnicity by using χ2 tests. We used multivariable logistic regression to assess associations between race/ethnicity and ICU admission. We used a post hoc analysis to assess associations between ICU admission and obstetric outcomes by race/ethnicity. RESULTS: After adjustment, we found a significantly higher ICU admission rate among Asian (adjusted odds ratio [aOR] = 1.30; 95% CI, 1.04-1.62; P = .02), Filipino (aOR = 1.45; 95% CI, 1.17-1.79; P < .001), and Native Hawaiian/Other Pacific Islander (aOR = 1.39; 95% CI, 1.15-1.68; P < .001) women compared with non-Hispanic White women. Multiple clinical characteristics and outcomes were associated with ICU admission, such as preexisting chronic conditions and pregnancy-induced hypertensive disorders. CONCLUSION: We found that severe maternal morbidity represented by ICU admission is higher among Asian, Filipino, and Native Hawaiian/Other Pacific Islander women than among non-Hispanic White women in Hawai'i. Our findings reemphasize the need for health care providers to be vigilant in caring for members of racial/ethnic minority groups and managing their comorbidities.

The structure of the mammalian RNase H2 complex provides insight into RNA.NA hybrid processing to prevent immune dysfunction.

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.

Evaluation of copper-1,3,5-benzenetricarboxylate metal-organic framework (Cu-MOF) as a selective sorbent for Lewis-base analytes.

The metal-organic framework copper-1,3,5-benzenetricarboxylate (Cu-BTC) was evaluated for its ability to selectively interact with Lewis-base analytes by examining retention on gas-chromatographic columns packed with Chromosorb W HP that contained 3.0% SE-30 along with various loadings of Cu-BTC. Scanning electron microscopy images of the support material showed the characteristic Cu-BTC crystals embedded in the SE-30 coating on the diatomaceous support. The results indicated that the Cu-BTC-containing stationary phase had limited thermal stability (220 °C) and strong general retention for analytes. Kováts index calculations showed selective retention (amounting to about 300 Kováts units) relative to n-alkanes for many small Lewis-base analytes on a column that contained 0.75% Cu-BTC compared with an SE-30 control. Short columns that contained lower loadings of Cu-BTC (0.10%) allowed elution of nitroaromatics; however, selectivity was not observed for aromatic compounds (including nitroaromatics) or nitroalkanes. Observed retention characteristics are discussed.

RNaseH2 mutants that cause Aicardi-Goutieres syndrome are active nucleases.

Mutations in the genes encoding the RNaseH2 and TREX1 nucleases have been identified in patients with Aicardi-Goutieres syndrome (AGS). To determine if the AGS RNaseH2 mutations result in the loss of nuclease activity, the human wild-type RNaseH2 and four mutant complexes that constitute the majority of mutations identified in AGS patients have been prepared and tested for ribonuclease H activity. The heterotrimeric structures of the mutant RNaseH2 complexes are intact. Furthermore, the ribonuclease H activities of the mutant complexes are indistinguishable from the wild-type enzyme with the exception of the RNaseH2 subunit A (Gly37Ser) mutant, which exhibits some evidence of altered nuclease specificity. These data indicate that the mechanism of RNaseH2 dysfunction in AGS cannot be simply explained by loss of ribonuclease H activity and points to a more complex mechanism perhaps mediated through altered interactions with as yet identified nucleic acids or protein partners.

Highly-Effective Contraception Use More Likely Among Native Hawaiian Women than Non-Native Hawaiian Women at Title X Clinics in Hawai'i.

Differences in contraceptive method use have been noted among women of different races, but studies describing contraceptive method use among Native Hawaiian women have not been published. To examine method choice in this group, the authors conducted a database review of the Hawai'i State Department of Health Title X program. Reviewed were client visit records (CVRs) that health care providers completed for women who were ages 15-44 years, avoiding pregnancy, not currently pregnant, and using a contraceptive method (N=54 513). Because a patient could have had several visits during the study period, the contraceptive method chosen at the last visit was selected for analysis. Statistical analyses included descriptive statistics, bivariate analyses, and logistic regression. The proportion of Native Hawaiian women who selected a highly-effective method of contraception (HEC), defined as an intrauterine device, implant, or permanent contraception, was higher than the proportion of non-Native Hawaiian women who selected an HEC. Overall, 15.4% of Native Hawaiian women during the study period chose HEC, compared to 8.8% of non-Native Hawaiian women. In a logistic regression analysis, Native Hawaiian women ages 15-29 were 1.46 times more likely to use HEC (95% CI: 1.35-1.58) than non-Native Hawaiian women ages 15-29, and Native Hawaiian women ages 30-44 were 1.69 times more likely to use HEC (95% CI: 1.53-1.87) than non-Native Hawaiian women in the same age group. Because Native Hawaiian women are reported to have higher rates of unintended pregnancy in the state compared to other racial groups, additional research exploring contraceptive non-use and pregnancy intention are needed.

A fight-and-flight for life: A rare case of advanced cervical cancer in pregnancy.

BACKGROUND: Advanced cervical cancer during pregnancy is an extremely rare event. We describe a case of at least stage IIIB cervical squamous cell carcinoma during pregnancy. This may possibly represent the longest gestation from time of diagnosis to delivery in a case of advanced cervical cancer, with potentially the most advanced gestational age at delivery and a relatively favorable outcome in the current literature.Case: A 29-year-old female at 20 0/7 weeks of gestation with at least stage IIIB squamous cell carcinoma of the cervix flew from Micronesia to Hawaii for oncologic treatment. After consultation with gynecologic oncology and maternal-fetal medicine, she opted to continue the pregnancy and began neoadjuvant chemotherapy with carboplatin and paclitaxel. At 33 2/7 weeks of gestation, she was admitted for preterm prelabor rupture of membranes and immediately underwent a cesarean delivery for heavy vaginal bleeding. Postpartum, she underwent cisplatin chemotherapy with concurrent radiation therapy. After 6 cycles of chemotherapy, the patient's cancer had progressed to the point that hospice was recommended. She died 11 months after initial presentation. CONCLUSION: Advanced cervical cancer during pregnancy requires individualized treatment, shared decision making, and a multidisciplinary team approach. If the pregnancy is continued, antepartum chemotherapy should be strongly considered. Maternal prognoses tend to be poor, but neonatal outcomes appear to be favorable.