Phase separation drives aberrant chromatin looping and cancer development.

The development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human haematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains the nucleoporin IDR-tandemly dispersed repeats of phenylalanine and glycine residues1,2. However, how unstructured IDRs contribute to oncogenesis remains unclear. Here we show that IDRs contained within NUP98-HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukaemias1,2, are essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukaemic transformation. Notably, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad 'super-enhancer'-like binding pattern typically seen at leukaemogenic genes, which potentiates transcriptional activation. An artificial HOX chimera, created by replacing the phenylalanine and glycine repeats of NUP98 with an unrelated LLPS-forming IDR of the FUS protein3,4, had similar enhancing effects on the genome-wide binding and target gene activation of the chimera. Deeply sequenced Hi-C revealed that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin loops that are enriched at proto-oncogenes. Together, this report describes a proof-of-principle example in which cancer acquires mutation to establish oncogenic transcription factor condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumourous transformation. As LLPS-competent molecules are frequently implicated in diseases1,2,4-7, this mechanism can potentially be generalized to many malignant and pathological settings.

ZFX Mediates Non-canonical Oncogenic Functions of the Androgen Receptor Splice Variant 7 in Castrate-Resistant Prostate Cancer.

Androgen receptor splice variant 7 (AR-V7) is crucial for prostate cancer progression and therapeutic resistance. We show that, independent of ligand, AR-V7 binds both androgen-responsive elements (AREs) and non-canonical sites distinct from full-length AR (AR-FL) targets. Consequently, AR-V7 not only recapitulates AR-FL's partial functions but also regulates an additional gene expression program uniquely via binding to gene promoters rather than ARE enhancers. AR-V7 binding and AR-V7-mediated activation at these unique targets do not require FOXA1 but rely on ZFX and BRD4. Knockdown of ZFX or select unique targets of AR-V7/ZFX, or BRD4 inhibition, suppresses growth of castration-resistant prostate cancer cells. We also define an AR-V7 direct target gene signature that correlates with AR-V7 expression in primary tumors, differentiates metastatic prostate cancer from normal, and predicts poor prognosis. Thus, AR-V7 has both ARE/FOXA1 canonical and ZFX-directed non-canonical regulatory functions in the evolution of anti-androgen therapeutic resistance, providing information to guide effective therapeutic strategies.

Alternative splicing regulation of membrane trafficking genes during myogenesis.

Alternative splicing transitions occur during organ development, and, in numerous diseases, splicing programs revert to fetal isoform expression. We previously found that extensive splicing changes occur during postnatal mouse heart development in genes encoding proteins involved in vesicle-mediated trafficking. However, the regulatory mechanisms of this splicing-trafficking network are unknown. Here, we found that membrane trafficking genes are alternatively spliced in a tissue-specific manner, with striated muscles exhibiting the highest levels of alternative exon inclusion. Treatment of differentiated muscle cells with chromatin-modifying drugs altered exon inclusion in muscle cells. Examination of several RNA-binding proteins revealed that the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking regulate splicing of trafficking genes during myogenesis, and that removal of PTBP1 motifs prevented PTBP1 from binding its RNA target. These findings enhance our understanding of developmental splicing regulation of membrane trafficking proteins which might have implications for muscle disease pathogenesis.

Upregulated microRNA-125b-5p in patients with asthma-COPD overlap mediates oxidative stress and late apoptosis via targeting IL6R/TRIAP1 signaling.

BACKGROUND: Among patients with chronic obstructive pulmonary disease (COPD), some have features of both asthma and COPD-a condition categorized as asthma-COPD overlap (ACO). Our aim was to determine whether asthma- or COPD-related microRNAs (miRNAs) play a role in the pathogenesis of ACO. METHODS: A total of 22 healthy subjects and 27 patients with ACO were enrolled. We selected 6 miRNAs that were found to correlate with COPD and asthma. The expression of miRNAs and target genes was analyzed using quantitative reverse-transcriptase polymerase chain reaction. Cell apoptosis and intracellular reactive oxygen species production were evaluated using flow cytometry. In vitro human monocytic THP-1 cells and primary normal human bronchial epithelial (NHBE) cells under stimuli with cigarette smoke extract (CSE) or ovalbumin (OVA) allergen or both were used to verify the clinical findings. RESULTS: We identified the upregulation of miR-125b-5p in patients with ACO and in THP-1 cells stimulated with CSE plus OVA allergen. We selected 16 genes related to the miR-125b-5p pathway and found that IL6R and TRIAP1 were both downregulated in patients with ACO and in THP-1 cells stimulated with CSE plus OVA. The percentage of late apoptotic cells increased in the THP-1 cell culture model when stimulated with CSE plus OVA, and the effect was reversed by transfection with miR-125b-5p small interfering RNA (siRNA). The percentage of reactive oxygen species-producing cells increased in the NHBE cell culture model when stimulated with CSE plus OVA, and the effect was reversed by transfection with miR-125b-5p siRNA. In NHBE cells, siRNA transfection reversed the upregulation of STAT3 under CSE+OVA stimulation. CONCLUSIONS: Our study revealed that upregulation of miR-125b-5p in patients with ACO mediated late apoptosis in THP-1 cells and oxidative stress in NHBE cells via targeting IL6R and TRIAP1. STAT3 expression was also regulated by miR-125b-5p.

LIGHT (TNFSF14) Costimulation Enhances Myeloid Cell Activation and Antitumor Immunity in the Setting of PD-1/PD-L1 and TIGIT Checkpoint Blockade.

Coinhibition of TIGIT (T cell immunoreceptor with Ig and ITIM domains) and PD-1/PD-L1 (PD-1/L1) may improve response rates compared with monotherapy PD-1/L1 blockade in checkpoint naive non-small cell lung cancer with PD-L1 expression >50%. TIGIT mAbs with an effector-competent Fc can induce myeloid cell activation, and some have demonstrated effector T cell depletion, which carries a clinical liability of unknown significance. TIGIT Ab blockade translates to antitumor activity by enabling PVR signaling through CD226 (DNAM-1), which can be directly inhibited by PD-1. Furthermore, DNAM-1 is downregulated on tumor-infiltrating lymphocytes (TILs) in advanced and checkpoint inhibition-resistant cancers. Therefore, broadening clinical responses from TIGIT blockade into PD-L1low or checkpoint inhibition-resistant tumors, may be induced by immune costimulation that operates independently from PD-1/L1 inhibition. TNFSF14 (LIGHT) was identified through genomic screens, in vitro functional analysis, and immune profiling of TILs as a TNF ligand that could provide broad immune activation. Accordingly, murine and human bifunctional fusion proteins were engineered linking the extracellular domain of TIGIT to the extracellular domain of LIGHT, yielding TIGIT-Fc-LIGHT. TIGIT competitively inhibited binding to all PVR ligands. LIGHT directly activated myeloid cells through interactions with LTßR (lymphotoxin ß receptor), without the requirement for a competent Fc domain to engage Fcγ receptors. LIGHT costimulated CD8+ T and NK cells through HVEM (herpes virus entry mediator A). Importantly, HVEM was more widely expressed than DNAM-1 on T memory stem cells and TILs across a range of tumor types. Taken together, the mechanisms of TIGIT-Fc-LIGHT promoted strong antitumor activity in preclinical tumor models of primary and acquired resistance to PD-1 blockade, suggesting that immune costimulation mediated by LIGHT may broaden the clinical utility of TIGIT blockade.

Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors.

Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency.

Behavioral and Psychological Factors in Buffering Diabetes-related Disability Development.

BACKGROUND: Diabetes is associated with disability development. Healthy behaviors and psychosocial support can help patients manage their disease. PURPOSE: To examine the role of various behavioral and psychological factors in buffering the effect of diabetes on disability development over time in Taiwanese adults. METHODS: Data on 5,131 adults aged ≥50 years were obtained from the Taiwan Longitudinal Study on Aging. A cohort sequential multilevel design was employed to analyze the association between behavioral and psychosocial factors and the risk of disability over a 11-year period. RESULTS: In patients with diabetes, having social support and exercising more than six times a week were associated with 4% and 49% reductions in the risk of disability, respectively (ßdiabetes*socialsupport = -0.285, p = .006; ßdiabetes*exercise3 = -2.612, p = .007). Exercising more than six times a week had an additional significant protective effect against disability development per year (ßdiabetes*exercises3*age = -0.241, p = .038). Depression did not significantly interact with diabetes. However, a trajectory analysis revealed that individuals who had both diabetes and depression had the highest disability score from middle age among all participants. CONCLUSIONS: Engaging in frequent exercise is the most influential factor for reducing the risk of disability in patients with diabetes. Social support provides an additional benefit for disability prevention in individuals with diabetes.

Diabetes can lead to several diseases that affect the heart, vessels, brain, kidneys, and nerves. These diseases can cause disabilities in people with diabetes. Disabilities among people with diabetes lead to higher death rates, depression, and poor life quality. All these have become a great burden to our public health care system. Several past studies let us know healthy behaviors and good social support have positive effects on reducing complications of diabetes. Healthy behaviors, such as healthy diets, regular exercises, and proper stress management. Good social support and participation are known to reduce dementia in people with diabetes. However, it remains unclear about interactions and exact associations among all these factors. This study was designed to find out how health behaviors and social support help people with diabetes reduce the risks of disabilities. By analyzing data from Taiwan Longitudinal Study on Aging, we found that exercising more than six times a week reduced disability risk the most for people with diabetes. Social support, although not as effective as exercising, provided additional positive effects to reducing disability risks of people with diabetes.

Impact of Coexisting Risk Factors on Outcomes in Patients with Acute Coronary Syndrome: A Real-World Analysis Using the Taiwan Chang Gung Research Database.

BACKGROUND Diabetes mellitus (DM), chronic kidney disease (CKD), and advanced age are associated with poor outcomes in patients with acute coronary syndrome (ACS). This real-world study utilized data from the Taiwan Chang Gung Research Database (CGRD) to compare outcomes in ACS patients with DM, CKD, and the elderly. MATERIAL AND METHODS The study enrolled 28,613 ACS patients diagnosed based on CGRD medical records between January 2005 and December 2019. Baseline characteristics and clinical outcomes were compared among groups based on patient characteristics. RESULTS Within the ACS cohort, 42.1% had DM, 48.2% had CKD, and 33.6% were elderly. Among them, 10.7% (3,070) were elderly patients with both DM and CKD. Elderly patients with DM and CKD had significantly higher risks of gastrointestinal bleeding (hazard ratio=11.32), cardiovascular events (HR=7.29), and all-cause mortality (HR=8.59). Patients with three or at least two of these risk factors had a 2.20-2.99-fold increased risk of recurrent ACS during the three-year follow-up period. CONCLUSIONS Patients with the combination of DM, CKD, and advanced age (elderly) experienced an 11.32-fold increased risk of gastrointestinal bleeding, 7.29-fold increased risk of cardiovascular events, and 8.59-fold increased risk of all-cause mortality compared to those without these risk factors. Furthermore, patients with two or more of these risk factors had a 2- to 3-fold increased risk of recurrent ACS. These findings emphasize the importance of managing multiple risk factors in ACS patients to improve outcomes.

A conserved BAH module within mammalian BAHD1 connects H3K27me3 to Polycomb gene silencing.

Trimethylation of histone H3 lysine 27 (H3K27me3) is important for gene silencing and imprinting, (epi)genome organization and organismal development. In a prevalent model, the functional readout of H3K27me3 in mammalian cells is achieved through the H3K27me3-recognizing chromodomain harbored within the chromobox (CBX) component of canonical Polycomb repressive complex 1 (cPRC1), which induces chromatin compaction and gene repression. Here, we report that binding of H3K27me3 by a Bromo Adjacent Homology (BAH) domain harbored within BAH domain-containing protein 1 (BAHD1) is required for overall BAHD1 targeting to chromatin and for optimal repression of the H3K27me3-demarcated genes in mammalian cells. Disruption of direct interaction between BAHD1BAH and H3K27me3 by point mutagenesis leads to chromatin remodeling, notably, increased histone acetylation, at its Polycomb gene targets. Mice carrying an H3K27me3-interaction-defective mutation of Bahd1BAH causes marked embryonic lethality, showing a requirement of this pathway for normal development. Altogether, this work demonstrates an H3K27me3-initiated signaling cascade that operates through a conserved BAH 'reader' module within BAHD1 in mammals.

Cistrome analysis of YY1 uncovers a regulatory axis of YY1:BRD2/4-PFKP during tumorigenesis of advanced prostate cancer.

Castration-resistant prostate cancer (CRPC) is a terminal disease and the molecular underpinnings of CRPC development need to be better understood in order to improve its treatment. Here, we report that a transcription factor Yin Yang 1 (YY1) is significantly overexpressed during prostate cancer progression. Functional and cistrome studies of YY1 uncover its roles in promoting prostate oncogenesis in vitro and in vivo, as well as sustaining tumor metabolism including the Warburg effect and mitochondria respiration. Additionally, our integrated genomics and interactome profiling in prostate tumor show that YY1 and bromodomain-containing proteins (BRD2/4) co-occupy a majority of gene-regulatory elements, coactivating downstream targets. Via gene loss-of-function and rescue studies and mutagenesis of YY1-bound cis-elements, we unveil an oncogenic pathway in which YY1 directly binds and activates PFKP, a gene encoding the rate-limiting enzyme for glycolysis, significantly contributing to the YY1-enforced Warburg effect and malignant growth. Altogether, this study supports a master regulator role for YY1 in prostate tumorigenesis and reveals a YY1:BRD2/4-PFKP axis operating in advanced prostate cancer with implications for therapy.

The Relationship of Conduction Disorder and Prognosis in Patients with Acute Coronary Syndrome.

Objective: Conduction disorders with a widened QRS are associated with poor prognosis in patients with acute coronary syndrome (ACS). Conduction disorders include left bundle branch block (LBBB), right bundle branch block (RBBB), and nonspecific intraventricular conduction delay (NICD). Previous studies did not have conflicting results regarding the type of bundle branch block (BBB) with the worst prognosis, and few studies have focused on the prognosis of patients with NICD. Methods: Patients with ACS were enrolled between January 2005 and December 2019, and their medical history (International Classification of Diseases codes) was obtained from the Chang Gung Research Database. Age, sex, comorbidities, left ventricular ejection fraction (LVEF), and drug use were compared between the patients with and without conduction disorders. The following clinical outcomes were compared between patients with and without conduction disorders: heart failure (HF) hospitalization, cardiovascular (CV) mortality, and all-cause mortality. After propensity score matching, the Kaplan-Meier curve analysis for HF hospitalization, CV mortality, and all-cause mortality were compared among patients with LBBB, RBBB, and NICD. Results: This study enrolled a total of 33970 participants and involved 3392 and 30578 patients with and without conduction disorders, respectively. Older age and a higher prevalence of comorbidities were noted in patients with conduction disorders. Lower mean LVEF was exhibited in the patients with conduction disorders (with vs. without; 44.64 ± 20.73% vs. 49.85 ± 20.63%; p < 0.001). During the 3-year follow-up period, higher incidences of HF hospitalization (21.55% vs. 17.51%; p < 0.001), CV mortality (17.98% vs. 12.14%; p < 0.001), and all-cause mortality (38.86% vs. 31.15%; p < 0.001) were noted in the patients with conduction disorder. After ACS events, 10.0% of patients presented with conduction disorders, with LBBB in 3.3%, RBBB in 6.0%, and NICD in 0.7%. The lowest mean of LVEF was presented in the patients with NICD (LBBB vs. RBBB vs. NICD; 41.00 ± 19.47% vs. 47.73 ± 20.82% vs. 34.57 ± 20.02%; p < 0.001). Among the three groups, the highest incidence of HF hospitalization was noted in patients with LBBB after propensity score matching. The lowest incidence of CV and all-cause mortality was observed in patients with RBBB. After adjustment of age, gender, comorbidities, medication, and mean LVEF, those with LBBB had the highest hazard ratio for major adverse cardiovascular events (MACEs) of 1.113 (p=0.029; 95% CI = 1.013-1.266). Conclusions: In the ACS population, patients with conduction delay had a poor prognosis due to a higher prevalence of comorbidities and lower mean LVEF. Among the patients with LBBB, RBBB, and NICD, those with LBBB and NICD had a higher incidence of HF hospitalization, CV mortality, and all-cause mortality. Patients with NICD had the lowest mean LVEF compared to those with LBBB and RBBB. Patients with LBBB had a significantly highest HR of MACE.

Genetic Deletion of HLJ1 Does Not Affect Blood Coagulation in Mice.

HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.

PHF19 promotes multiple myeloma tumorigenicity through PRC2 activation and broad H3K27me3 domain formation.

Dysregulation of polycomb repressive complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing trimethylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19; also known as polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. Chromatin immunoprecipitation followed by sequencing profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains, possibly due to impaired H3K27me3 spreading from cytosine guanine dinucleotide islands, which is reminiscent to the reported effect of an "onco"-histone mutation, H3K27 to methionine (H3K27M). RNA-sequencing-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Correlation studies using patient sample data sets further support a clinical relevance of the PHF19-regulated pathways. Lastly, we show that MM cells are generally sensitive to PRC2 inhibitors. Collectively, this study demonstrates that PHF19 promotes MM tumorigenesis through enhancing H3K27me3 deposition and PRC2's gene-regulatory functions, lending support for PRC2 blockade as a means for MM therapeutics.

Enterovirus A71 Containing Codon-Deoptimized VP1 and High-Fidelity Polymerase as Next-Generation Vaccine Candidate.

Enterovirus A71 (EV-A71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD), which occasionally results in severe neurological complications. In this study, we developed four EV-A71 (rgEV-A71) strains by reverse genetics procedures as possible vaccine candidates. The four rgEV-A71 viruses contained various codon-deoptimized VP1 capsid proteins (VP1-CD) and showed replication rates and antigenicity similar to that of the wild-type virus, while a fifth virus, rg4643C4VP-CD, was unable to form plaques but was still able to be examined by median tissue culture infectious dose (TCID50) titers, which were similar to those of the others, indicating the effect of CD on plaque formation. However, the genome stability showed that there were some mutations which appeared during just one passage of the VP1-CD viruses. Thus, we further constructed VP1-CD rgEV-A71 containing high-fidelity determinants in 3D polymerase (CD-HF), and the number of mutations in CD-HF rgEV-A71 was shown to have decreased. The CD-HF viruses showed less virulence than the parental strain in a mouse infection model. After 14 days postimmunization, antibody titers had increased in mice infected with CD-HF viruses. The mouse antisera showed similar neutralizing antibody titers against various CD-HF viruses and different genotypes of EV-A71. The study demonstrates the proof of concept that VP1 codon deoptimization combined with high-fidelity 3D polymerase decreased EV-A71 mutations and virulence in mice but retained their antigenicity, indicating it is a good candidate for next-generation EV-A71 vaccine development.IMPORTANCE EV-A71 can cause severe neurological diseases with fatality in infants and young children, but there are still no effective drugs to date. Here, we developed a novel vaccine strategy with the combination of CD and HF substitutions to generate the genetically stable reverse genetics virus. We found that CD combined with HF polymerase decreased the virulence but maintained the antigenicity of the virus. This work demonstrated the simultaneous introduction of CD genome sequences and HF substitutions as a potential new strategy to develop attenuated vaccine seed virus. Our work provides insight into the development of a low-virulence candidate vaccine virus through a series of genetic editing of virus sequences while maintaining its antigenicity and genome stability, which will provide an additional strategy for next-generation vaccine development of EV-A71.

Design, synthesis and evaluation of novel levoglucosenone derivatives as promising anticancer agents.

A series of levoglucosenone-derived 1,2,3-triazoles and isoxazoles featuring a flexible spacer between the heteroaromatic and anhydropyranose cores have been designed and synthesized following an hetero Michael // 1,3-dipolar cycloaddition path. The use of a design of experiments approach allowed the optimization of the oxa-Michael reaction with propargyl alcohol as nucleophile, a key step for the synthesis of the target compounds. All of the compounds were tested for their anticancer activity on MDA-MB-231 cells, featuring mutant p53. The results highlighted the importance of the introduction of the flexible spacer as well as the higher activity of oxa-Michael isoxazole-derivatives. The most prominent compounds also showed anti-proliferative activities against lung and colon cancer cell lines. The compounds showed enhanced cytotoxic effects in the presence of mutant p53, determined both by endogenous mutant p53 knock down (R280K) and by reintroducing p53 R280K in cells lacking p53 expression.

A Functional riboSNitch in the 3' Untranslated Region of FKBP5 Alters MicroRNA-320a Binding Efficiency and Mediates Vulnerability to Chronic Post-Traumatic Pain.

Previous studies have shown that common variants of the gene coding for FK506-binding protein 51 (FKBP5), a critical regulator of glucocorticoid sensitivity, affect vulnerability to stress-related disorders. In a previous report, FKBP5 rs1360780 was identified as a functional variant because of its effect on gene methylation. Here we report evidence for a novel functional FKBP5 allele, rs3800373. This study assessed the association between rs3800373 and post-traumatic chronic pain in 1607 women and men from two ethnically diverse human cohorts. The molecular mechanism through which rs3800373 affects adverse outcomes was established via in silico, in vivo, and in vitro analyses. The rs3800373 minor allele predicted worse adverse outcomes after trauma exposure, such that individuals with the minor (risk) allele developed more severe post-traumatic chronic musculoskeletal pain. Among these individuals, peritraumatic circulating FKBP5 expression levels increased as cortisol and glucocorticoid receptor (NR3C1) mRNA levels increased, consistent with increased glucocorticoid resistance. Bioinformatic, in vitro, and mutational analyses indicate that the rs3800373 minor allele reduces the binding of a stress- and pain-associated microRNA, miR-320a, to FKBP5 via altering the FKBP5 mRNA 3'UTR secondary structure (i.e., is a riboSNitch). This results in relatively greater FKBP5 translation, unchecked by miR-320a. Overall, these results identify an important gene-miRNA interaction influencing chronic pain risk in vulnerable individuals and suggest that exogenous methods to achieve targeted reduction in poststress FKBP5 mRNA expression may constitute useful therapeutic strategies.SIGNIFICANCE STATEMENTFKBP5 is a critical regulator of the stress response. Previous studies have shown that dysregulation of the expression of this gene plays a role in the pathogenesis of chronic pain development as well as a number of comorbid neuropsychiatric disorders. In the current study, we identified a functional allele (rs3800373) in the 3'UTR of FKBP5 that influences vulnerability to chronic post-traumatic pain in two ethnic cohorts. Using multiple complementary experimental approaches, we show that the FKBP5 rs3800373 minor allele alters the secondary structure of FKBP5 mRNA, decreasing the binding of a stress- and pain-associated microRNA, miR-320a. This results in relatively greater FKBP5 translation, unchecked by miR-320a, increasing glucocorticoid resistance and increasing vulnerability to post-traumatic pain.

Mononuclear Manganese(III) Superoxo Complexes: Synthesis, Characterization, and Reactivity.

Metal-superoxo species are typically proposed as key intermediates in the catalytic cycle of dioxygen activation by metalloenzymes involving different transition metal cofactors. In this regard, while a series of Fe-, Co-, and Ni-superoxo complexes have been reported to date, well-defined Mn-superoxo complexes remain rather rare. Herein, we report two mononuclear MnIII-superoxo species, Mn(BDPP)(O2•-) (2, H2BDPP = 2,6-bis((2-(S)-diphenylhydroxylmethyl-1-pyrrolidinyl)methyl)pyridine) and Mn(BDPBrP)(O2•-) (2', H2BDPBrP = 2,6-bis((2-(S)-di(4-bromo)phenylhydroxyl-methyl-1-pyrrolidinyl)methyl)pyridine), synthesized by bubbling O2 into solutions of their MnII precursors, Mn(BDPP) (1) and Mn(BDPBrP) (1'), at -80 °C. A combined spectroscopic (resonance Raman and electron paramagnetic resonance (EPR) spectroscopy) and computational study evidence that both complexes contain a high-spin MnIII center (SMn = 2) antiferromagnetically coupled to a superoxo radical ligand (SOO• = 1/2), yielding an overall S = 3/2 ground state. Complexes 2 and 2' were shown to be capable of abstracting a H atom from 2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPO-H) to form MnIII-hydroperoxo species, Mn(BDPP)(OOH) (5) and Mn(BDPBrP)(OOH) (5'). Complexes 5 and 5' can be independently prepared by the reactions of the isolated MnIII-aqua complexes, [Mn(BDPP)(H2O)]OTf (6) and [Mn(BDPBrP)(H2O)]OTf (6'), with H2O2 in the presence of NEt3. The parallel-mode EPR measurements established a high-spin S = 2 ground state for 5 and 5'.

Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly.

The exon junction complex (EJC) is an RNA binding complex comprised of the core components Magoh, Rbm8a, and Eif4a3. Human mutations in EJC components cause neurodevelopmental pathologies. Further, mice heterozygous for either Magoh or Rbm8a exhibit aberrant neurogenesis and microcephaly. Yet despite the requirement of these genes for neurodevelopment, the pathogenic mechanisms linking EJC dysfunction to microcephaly remain poorly understood. Here we employ mouse genetics, transcriptomic and proteomic analyses to demonstrate that haploinsufficiency for each of the 3 core EJC components causes microcephaly via converging regulation of p53 signaling. Using a new conditional allele, we first show that Eif4a3 haploinsufficiency phenocopies aberrant neurogenesis and microcephaly of Magoh and Rbm8a mutant mice. Transcriptomic and proteomic analyses of embryonic brains at the onset of neurogenesis identifies common pathways altered in each of the 3 EJC mutants, including ribosome, proteasome, and p53 signaling components. We further demonstrate all 3 mutants exhibit defective splicing of RNA regulatory proteins, implying an EJC dependent RNA regulatory network that fine-tunes gene expression. Finally, we show that genetic ablation of one downstream pathway, p53, significantly rescues microcephaly of all 3 EJC mutants. This implicates p53 activation as a major node of neurodevelopmental pathogenesis following EJC impairment. Altogether our study reveals new mechanisms to help explain how EJC mutations influence neurogenesis and underlie neurodevelopmental disease.

Meta-analysis of airway epithelium gene expression in asthma.

Differential gene expression in the airway epithelium of patients with asthma versus controls has been reported in several studies. However, there is no consensus on which genes are reproducibly affected in asthma. We sought to identify a consensus list of differentially expressed genes (DEGs) using a meta-analysis approach.We identified eight studies with data that met defined inclusion criteria. These studies comprised 355 cases and 193 controls and involved sampling either bronchial or nasal epithelium. We conducted study-level analyses, followed by a meta-analysis. Likewise, we applied a meta-analysis framework to the results of study-level pathway enrichment.We identified 1273 DEGs, 431 of which had not been identified in previous studies. 450 DEGs exhibited large effect sizes and were robust to study population differences in age, sex, race/ethnicity, medication use, smoking status and exacerbations. The magnitude of differential expression of these 450 genes was highly similar in bronchial and nasal airway epithelia. Meta-analysis of pathway enrichment revealed a number of consistently dysregulated biological pathways, including putative transcriptional and post-transcriptional regulators.In total, we identified a set of genes that is consistently dysregulated in asthma, that links to known and novel biological pathways, and that will inform asthma subtype identification.

Synthesis of Triazole Derivatives of Levoglucosenone As Promising Anticancer Agents: Effective Exploration of the Chemical Space through retro-aza-Michael//aza-Michael Isomerizations.

The design and synthesis of biomass-derived triazoles and the in vitro evaluation as potential anticancer agents are described. The discovery of base-catalyzed retro-aza-Michael//aza-Michael isomerizations allowed the exploration of the chemical space by affording novel types of triazoles, difficult to obtain otherwise. Following this strategy, 2,4-disubstituted 1,2,3-triazoles could be efficiently obtained from the corresponding 1,4-disubstituted analogues.