Using Experimental and Computational Strategies to Understand the Biogenesis of microRNAs and piRNAs: A Dissertation
Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies.
The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation.
In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.
Predictors and outcomes of readmission for Clostridium difficile in a national sample of medicare beneficiaries
BACKGROUND: Rates of Clostridium difficile (CD) infections are increasing. Elderly patients may be at particular risk of recurrent CD infection. Little is known about the risk for CD readmission specifically in this age group.
METHODS: A 5% random sample of Medicare data (2009-2011) was queried for patients surviving a hospitalization for CD by ICD-9 code. Demographic (age, sex, gender), clinical (Elixhauser index, gastrointestinal comorbidities), and hospitalization (length of stay, ICU admission) characteristics as well as exposure to antibiotics and interim non-CD hospitalization were compared for those with and without a readmission for CD. A multivariable survival analysis was used to determine predictors of readmission.
RESULTS: Of 7,564 patients surviving a CD hospitalization, 8.5% were readmitted with CD in a median of 25 days (interquartile range (IQR) 14-57). In multivariable survival analyses, interim non-CD hospital exposure was the strongest predictor of CD readmission (hazard ration (HR) 3.75 95%, confidence interval (CI) 3.2-4.42). Oral and intravenous/intramuscular (IV/IM) antibiotic use, Elixhauser index, and CD as the primary diagnosis also increased the risk of CD readmission. Discharge to hospice, long-term care or a skilled nursing facility decreased the odds of CD readmission.
CONCLUSION: Hospital exposure and antibiotic use put elderly patients at risk of CD readmission. Exposure to these factors should be minimized in the immediate post discharge period.
Understanding the behaviors of surrogate seekers (those who seek health information for others) may guide efforts to improve health information transmission. We used 2011-2012 data from the Health Information National Trends Survey to describe behaviors of online surrogate seekers. Respondents were asked about use of the Internet for surrogate-seeking over the prior 12 months. Data were weighted to calculate population estimates. Two thirds (66.6%) reported surrogate-seeking. Compared to those who sought health information online for only themselves, surrogate seekers were more likely to live in households with others (weighted percent 89.4 vs. 82.5% of self-seekers; p < 0.05); no significant differences in sex, race, income or education were observed. Surrogate seekers were more likely to report activities requiring user-generated content: email communication with healthcare providers; visits to social networking sites to read and share about medical topics and participation in online health support groups. On multivariate analysis, those who had looked online for healthcare providers were more likely to be surrogate seekers (OR 1.67, 95% CI 1.08-2.59). In addition to seeking health information, surrogate seekers create and pass along communications that may influence medical care decisions. Research is needed to identify ways to facilitate transmission of accurate health information.
INTRODUCTION: As a common monogenic disease, alpha-1 antitrypsin (AAT) deficiency has undergone thorough investigation for the development of gene therapy. The most common pathology associated with AAT deficiency occurs in the lung, where the loss of function due to impaired secretion of mutant AAT prevents the inhibition of neutrophil elastase and leads to loss of elastin content from the alveolar interstitium.
AREAS COVERED: Current treatment in the USA consists of recurrent intravenous protein replacement therapy to augment serum AAT levels. In an attempt to replace recurring treatments with a single dose of gene therapy, recombinant adenovirus, plasmid, and recombinant adeno-associated virus (rAAV) vectors have been investigated as vectors for transgene delivery.
EXPERT OPINION: Large strides in gene therapy for AAT deficiency lung disease have led to the development of rAAV1-AAT capable of producing sustained serum AAT levels in clinical trials after intramuscular administration in humans at 3% of the target level. Further increases in levels are anticipated as limb perfusion targets greater muscle mass. The future roles of intrapleural and airway delivery, miRNA-expressing vectors, iPS cell platforms, and genome editing are anticipated.
Reduced frequencies of polyfunctional CMV-specific T cell responses in infants with congenital CMV infection
PURPOSE: CMV infection remains a priority for vaccine development. Vaccination of infants could modify congenital infection and provide lifetime immunity. Properties of CMV-specific T cells associated with control of viral replication in early life have not been fully defined.
METHODS: CMV-specific CD4 and CD8 T cell responses were investigated in infants with congenital CMV infection and compared to adults with primary or chronic infection. PBMC were stimulated with UL83 (pp65) or UL122 (IE-2) peptide pools then stained with antibodies to markers of T cell subset (CD4 or CD8), phenotype (CD45RA, CCR7), or function (MIP1beta, CD107, IFNgamma, IL2) for flow cytometry analysis.
RESULTS: Detection of CMV pp65-specific CD4 T cells was less common in infants than adults. Responder cells were primarily effector memory (EM, CD45RA-CCR7-) in adults, but mixed memory subsets in infants. Detection of CMV pp65-specific CD8 T cells did not differ between the groups, but infants had lower frequencies of total responding cells and of MIP1beta- or CD107-expressing cells. Responder cells were EM or effector memory RA (CD45RA + CCR7-) in all groups. Polyfunctional T cells were less commonly detected in infants than adults. Responses to IE-2 were detected in adults but not infants. All infants had detectable circulating CMV DNA at initial study (versus 60 % of adults with primary infection) despite longer duration of CMV infection.
CONCLUSIONS: Reduced frequencies and altered functional profile of CMV-specific CD4 and CD8 T cell responses were detected in infants compared to adults, and were associated with persistent CMV DNA in peripheral blood.
OBJECTIVE: This article reviews what is known about behavioral health treatment of deaf persons with comorbid trauma and addiction.
METHOD: We discuss how to work therapeutically with deaf people with comorbid trauma and addiction, both through a review of the literature and through clinical observations of the authors. The article also includes the personal stories of two people-a Deaf peer specialist and a hearing psychiatrist-who share their humbling stories about the recovery process for deaf people and the challenges of learning to become an effective Deaf mental health care provider.
FINDINGS: Deaf people report higher rates of mental health problems than the general population. Although initial empirical work with the deaf population suggests high rates of posttraumatic stress disorder (PTSD) and substance use disorder (SUD), little is known about the rates of comorbid PTSD/SUD or how to effectively address this comorbidity in treatment.
CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Substantial work is needed to raise awareness of comorbid PTSD/SUD and provide treatment tools to agencies and providers who work with deaf clients, infusing trauma-informed care into deaf SUD services and SUD treatment into deaf mental health care. Fortunately, several endeavors are on the horizon to disseminate assessment and treatment tools to work with deaf people recovering from trauma and addiction.
Pregnancy intention, receipt of pre-conception care, and pre-conception weight counseling reported by overweight and obese women in late pregnancy
We surveyed overweight or obese women receiving prenatal care for a singleton pregnancy at a large academic medical center in 2010. During late pregnancy, women reported pregnancy intentionality and pre-conception weight counseling. Participants (N = 82) had a mean age of 29.7 (SD: 6.3) years, 61% were non-Hispanic white, 47% were nulliparous. Before pregnancy, 45% were overweight and 55% were obese. Forty-eight percent reported that the current pregnancy was planned. Of these women, 36% reported a pre-conception visit. Of these, 29% reported pre-conception weight counseling (5% of sample). Unrealized opportunities exist in the clinical setting for promoting weight management during the childbearing years.
Recombinant adeno-associated virus-mediated inhibition of microRNA-21 protects mice against the lethal schistosome infection by repressing both IL-13 and transforming growth factor beta 1 pathways
Schistosomiasis is a serious parasitic disease in humans, which can lead to liver fibrosis and death. Accumulating evidence indicated that targeting the deregulated microRNAs (miRNAs) could mitigate disease outcomes. Here, we showed that progressive hepatic schistosomiasis caused elevation of miR-21 and efficient and sustained inhibition of miR-21 by using highly hepatic tropic adeno-associated virus serotype 8 (rAAV8), which protected mice against lethal schistosome infection through attenuation of hepatic fibrosis (HF). We demonstrated an additive role of interleukin (IL)-13 and transforming growth factor beta 1 (TGF-beta1) in up-regulating miR-21 expression in hepatic stellate cells (HSCs) by activation of mothers against decapentaplegic (SMAD) proteins. Furthermore, down-regulation of miR-21 in HSCs reversed HF by enhancing SMAD7 expression, thus repressing TGF-beta1/Smad and IL-13/Smad pathways. CONCLUSION: This study suggests the mechanism of IL-13-mediated schistosomiasis HF by up-regulation of miR-21 and highlights the potential of rAAV8-mediated miR-21 inhibition as a therapeutic intervention for hepatic fibrotic diseases, such as schistosomiasis.
50 year trends in atrial fibrillation prevalence, incidence, risk factors, and mortality in the Framingham Heart Study: a cohort study
BACKGROUND: Comprehensive long-term data on atrial fibrillation trends in men and women are scant. We aimed to provide such data through analysis of the Framingham cohort over 50 years.
METHODS: We investigated trends in incidence, prevalence, and risk factors for atrial fibrillation and its association with stroke and mortality after onset in 9511 participants enrolled in the Framingham Heart Study between 1958 and 2007. We analysed trends within 10 year groups (1958-67, 1968-77, 1978-87, 1988-97, and 1998-2007), stratified by sex.
FINDINGS: During 50 years of observation (202,417 person-years), 1544 cases of new-onset atrial fibrillation occurred (of whom 723 [47%] were women). Between 1958-67 and 1998-2007, age-adjusted prevalence of atrial fibrillation quadrupled from 20.4 to 96.2 cases per 1000 person-years in men and from 13.7 to 49.4 cases per 1000 person-years in women; age-adjusted incidence increased from 3.7 to 13.4 new cases per 1000 person-years in men and from 2.5 to 8.6 new cases per 1000 person-years in women (ptrend < 0.0001 for all comparisons). For atrial fibrillation diagnosed by electrocardiograph (ECG) during routine Framingham examinations, age-adjusted prevalence per 1000 person-years increased (12.6 in 1958-67 to 25.7 in 1998-2007 in men, ptrend=0.0007; 8.1 to 11.8 in women, ptrend=0.009). However, age-adjusted incidence of atrial fibrillation by Framingham Heart Study ECGs did not change significantly with time. Although the prevalence of most risk factors changed over time, their associated hazards for atrial fibrillation changed little. Multivariable-adjusted proportional hazards models revealed a 74% (95% CI 50-86%) decrease in stroke (hazards ratio [HR] 3.77, 95% CI 1.98-7.20 in 1958-1967 compared with 1998-2007; ptrend=0.0001) and a 25% (95% CI -3-46%) decrease in mortality (HR 1.34, 95% CI 0.97-1.86 in 1958-1967 compared with 1998-2007; ptrend=0.003) in 20 years following atrial fibrillation onset.
INTERPRETATION: Trends of increased incidence and prevalence of atrial fibrillation in the community were probably partly due to enhanced surveillance. Measures are needed to enhance early detection of atrial fibrillation, through increased awareness coupled with targeted screening programmes and risk factor-specific prevention.
FUNDING: NIH, NHLBI, NINDS, Deutsche Forschungsgemeinschaft.
On the relative roles of background selection and genetic hitchhiking in shaping human cytomegalovirus genetic diversity
A central focus of population genetics has been examining the contribution of selective and neutral processes in shaping patterns of intraspecies diversity. In terms of selection specifically, surveys of higher organisms have shown considerable variation in the relative contributions of background selection and genetic hitchhiking in shaping the distribution of polymorphisms, though these analyses have rarely been extended to bacteria and viruses. Here, we study the evolution of a ubiquitous, viral pathogen, human cytomegalovirus (HCMV), by analyzing the relationship among intraspecies diversity, interspecies divergence, and rates of recombination. We show that there is a strong correlation between diversity and divergence, consistent with expectations of neutral evolution. However, after correcting for divergence, there remains a significant correlation between intraspecies diversity and recombination rates, with additional analyses suggesting that this correlation is largely due to the effects of background selection. In addition, a small number of loci, centered on long non-coding RNAs, also show evidence of selective sweeps. These data suggest that HCMV evolution is dominated by neutral mechanisms as well as background selection, expanding our understanding of linked selection to a novel class of organisms.
The impact of acute care surgery on appendicitis outcomes: Results from a national sample of university-affiliated hospitals
BACKGROUND: Acute appendicitis is the most common indication for emergency general surgery (EGS) in the United States. We examined the role of acute care surgery (ACS) on interventions and outcomes for acute appendicitis at a national sample of university-affiliated hospitals.
METHODS: We surveyed senior surgeons responsible for EGS coverage at University HealthSystems Consortium hospitals, representing more than 90% of university-affiliated hospitals in the United States. The survey elicited data on resources allocated for EGS during 2013. Responses were linked to University HealthSystems Consortium outcomes data by unique hospital identifiers. Patients treated at hospitals reporting hybrid models for EGS coverage were excluded. Differences in interventions and outcomes between patients with acute appendicitis treated at ACS hospitals versus hospitals with a general surgeon on-call model (GSOC) were analyzed using univariate comparisons and multivariable logistic regression models adjusted for patient demographics, clinical acuity, and hospital characteristics.
RESULTS: We found 122 hospitals meeting criteria for analysis where 2,565 patients were treated for acute appendicitis. Forty-eight percent of hospitals had an ACS model (n = 1,414), and 52% had a GSOC model (n = 1,151). Hospitals with ACS models were more likely to treat minority patients than those with GSOC models. Patients treated at ACS hospitals were more likely to undergo laparoscopic appendectomy. In multivariable modeling of patients who had surgery (n = 2,258), patients treated at ACS hospitals had 1.86 (95% confidence interval, 1.23-2.80) greater odds of undergoing laparoscopic appendectomy.
CONCLUSION: In an era when laparoscopic appendectomy is increasingly accepted for treating uncomplicated acute appendicitis, particularly in low-risk patients, it is concerning that patients treated at GSOC model hospitals are more likely to undergo traditional open surgery at the time of presentation. Furthermore, hospitals with ACS are functioning as safety-net hospitals for vulnerable patients with acute appendicitis.
LEVEL OF EVIDENCE: Therapeutic study, level IV.
This is the October 2015 issue of the UMass Center for Clinical and Translational Science Newsletter containing news and events of interest.
Enhancing Participation in Depression Care in Outpatient Perinatal Care Settings: A Systematic Review
OBJECTIVE: To examine a wide range of study designs and outcomes to estimate the extent to which interventions in outpatient perinatal care settings are associated with an increase in the uptake of depression care.
DATA SOURCES: PubMed, CINAHL, PsycINFO, ClinicalTrials.gov, and Scopus (EMBASE) were searched for studies published between 1999 and 2014 that evaluated mental health care use after screening for depression in perinatal care settings.
METHODS OF STUDY SELECTION: Inclusion criteria were: 1) English language; 2) pregnant and postpartum women who screened positive for depression; 3) exposure (validated depression screening in outpatient perinatal care setting); and, 4) outcome (mental health care use). Searches yielded 392 articles, 42 met criteria for full-text review, and 17 met inclusion criteria. Study quality was assessed using a modified Downs and Black scale.
TABULATION, INTEGRATION, AND RESULTS: Articles were independently reviewed by two abstractors and consensus reached. Study design, intervention components, and mental health care use were defined and categorized. Seventeen articles representing a range of study designs, including one randomized controlled trial and one cluster randomized controlled trial, were included. The average quality rating was 61% (31.0-90.0%). When no intervention was in place, an average of 22% (13.8-33.0%) of women who screened positive for depression had at least one mental health visit. The average rate of mental health care use was associated with a doubling of this rate with patient engagement strategies (44%, 29.0-90.0%), on-site assessments (49%, 25.2-90.0%), and perinatal care provider training (54%, 1.0-90.0%). High rates of mental health care use (81%, 72.0-90.0%) were associated with implementation of additional interventions, including resource provision to women, perinatal care provider training, on-site assessment, and access to mental health consultation for perinatal care providers.
CONCLUSION: Screening alone was associated with 22% mental health care use among women who screened positive for depression; however, implementation of additional interventions was associated with a two to fourfold increased use of mental health care. Although definitive studies are still needed, screening done in conjunction with interventions that target patient, health care provider, and practice-level barriers is associated with increased improved rates of depression detection, assessment, referral, and treatment in perinatal care settings.
Histone deacetylase 3 coordinates deacetylase-independent epigenetic silencing of TGFβ1 to orchestrate second heart field development
About two-thirds of human congenital heart disease (CHD) involves second heart field (SHF) derived structures. Histone-modifying enzymes, histone deacetylases (HDACs), regulate the epigenome; however, their functions within the second heart field remain elusive. Here we demonstrate that histone deacetylase 3 (Hdac3) orchestrates epigenetic silencing of Tgfβ1, a causative factor in CHD pathogenesis, in a deacetylase-independent manner to regulate development of SHF-derived structures. In murine embryos lacking Hdac3 in the SHF, increased Tgfβ1 bioavailability is associated with ascending aortic dilatation, outflow tract malrotation, overriding aorta, double outlet right ventricle, aberrant semilunar valve development, bicuspid aortic valve, ventricular septal defects, and embryonic lethality. Activation of Tgfβ signaling causes aberrant endothelial-to-mesenchymal transition (EndMT) and altered extracellular matrix homeostasis in Hdac3-null outflow tracts and semilunar valves and pharmacological inhibition of Tgfβ rescues these defects. Hdac3 recruits components of PRC2 complex, methyltransferase Ezh2, Eed, and Suz12 to the Ncor complex to enrich trimethylation of lys27 on histone H3 at the Tgfβ1 regulatory region and thereby maintains epigenetic silencing of Tgfβ1 specifically within the SHF-derived mesenchyme. Wild-type Hdac3 or catalytically-inactive Hdac3 expression rescue aberrant EndMT and epigenetic silencing of Tgfβ1 in Hdac3-null outflow tracts and semilunar valves. These findings reveal that epigenetic dysregulation within the SHF is a predisposing factor for CHD.
Data from: The Cardiovascular Effects of Adjunctive Metformin Therapy in Overweight/obese Youth with Type 1 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial
This dataset is the primary data source for a manuscript submitted for publication.
Context: The cardiovascular effect of adjunctive metformin therapy in overweight/obese youth with type 1 diabetes (T1D) is unknown.
Objective: To compare the effect of prolonged, adjunctive metformin vs. placebo therapy on markers of cardiovascular risk in overweight/obese youth with T1D based on differences in total cholesterol (TC)/ high-density lipoprotein (HDL) ratio, triglycerides (TG)/HDL ratio, Atherogenic Index of Plasma (AIP) log [TG/HDL] ratio, adiponectin/leptin ratio, and 25-hydroxyvitamin D [25(OH)D] concentration.
Hypothesis: Adjunctive metformin therapy will improve markers of cardiovascular health in overweight/obese youth with T1D.
Setting: University outpatient facility.
Design and Participants: A 9-mo randomized, double-blind, placebo-controlled trial of metformin (1000 mg daily) and placebo in 28 subjects (13m/15f) of ages 10-20years (y), with HbA1c >8%, BMI >85%, and T1D > 12 months. The metformin group consisted of 15 subjects (8 m/7f), of age 15.0±2.5 y; while the control group consisted of 13 subjects (5m/8f), of age 14.5±3.1y. Participants employed a self-directed treat-to-target insulin regimen based on a titration algorithm of (-2)-0-(+2) units to adjust long-acting insulin dose every 3rd day from -3 mo through +9 mo to maintain fasting plasma glucose between 90-120 mg/dL.
Results: After adjusting for age, gender, BMI, and baseline values, the metformin group had a clinically significant reduction in TC/HDL of 0.5 unit: 3.5[3.0-4.1] vs. 4.0 [3.3-4.4] (p=0.578); and TG/HDL of 1.0 unit, 2.6 [1.1-4.3] vs. 3.6 [2.0-5.2] (p=0.476); and AIP of 0.44 unit: -0.23 ± 0.9 vs. 0.21 ± 0.8 (p=0.251). Conversely, the metformin group had a clinically significant elevation in adiponectin/leptin ratio of 0.8 unit: 2.0[0.84-3.2] vs. 1.2[0.11-2.3], (p=0.057); and a mean serum 25(OH)D in the vitamin D sufficiency range, 31.3 ng/mL [22.3-40.4] compared to the placebo group's lower mean 25(OH)D of 25.8 ng/mL [14.1-35.9], (p=0.337).
Conclusions: Prolonged adjunctive metformin therapy may be cardio-protective in overweight/obese youth with T1D.
High-throughput sequencing has enabled many powerful approaches in biological research. Here, we review sequencing approaches to measure frequency changes within engineered mutational libraries subject to selection. These analyses can provide direct estimates of biochemical and fitness effects for all individual mutations across entire genes (and likely compact genomes in the near future) in genetically tractable systems such as microbes, viruses, and mammalian cells. The effects of mutations on experimental fitness can be assessed using sequencing to monitor time-dependent changes in mutant frequency during bulk competitions. The impact of mutations on biochemical functions can be determined using reporters or other means of separating variants based on individual activities (e.g., binding affinity for a partner molecule can be interrogated using surface display of libraries of mutant proteins and isolation of bound and unbound populations). The comprehensive investigation of mutant effects on both biochemical function and experimental fitness provide promising new avenues to investigate the connections between biochemistry, cell physiology, and evolution. We summarize recent findings from systematic mutational analyses; describe how they relate to a field rich in both theory and experimentation; and highlight how they may contribute to ongoing and future research into protein structure-function relationships, systems-level descriptions of cell physiology, and population-genetic inferences on the relative contributions of selection and drift.
This review discusses three inter-related topics: (1) the immaturity of the neonatal and infant immune response; (2) heterologous immunity, where prior infection history with unrelated pathogens alters disease outcome resulting in either enhanced protective immunity or increased immunopathology to new infections, and (3) epidemiological human vaccine studies that demonstrate vaccines can have beneficial or detrimental effects on subsequent unrelated infections. The results from the epidemiological and heterologous immunity studies suggest that the immune system has tremendous plasticity and that each new infection or vaccine that an individual is exposed to during a lifetime will potentially alter the dynamics of their immune system. It also suggests that each new infection or vaccine that an infant receives is not only perturbing the immune system but is educating the immune system and laying down the foundation for all subsequent responses. This leads to the question, is there an optimum way to educate the immune system? Should this be taken into consideration in our vaccination protocols?
PURPOSE OF REVIEW: beta Cells represent one of many cell types in heterogeneous pancreatic islets and play the central role in maintaining glucose homeostasis, such that disrupting beta-cell function leads to diabetes. This review summarizes the methods for isolating and characterizing beta cells, and describes integrated 'omics' approaches used to define the beta cell by its transcriptome and proteome.
RECENT FINDINGS: RNA sequencing and mass spectrometry-based protein identification have now identified RNA and protein profiles for mouse and human pancreatic islets and beta cells, and for beta-cell lines. Recent publications have outlined these profiles and, more importantly, have begun to assign the presence or absence of specific genes and regulatory molecules to beta-cell function and dysfunction. Overall, researchers have focused on understanding the pathophysiology of diabetes by connecting genome, transcriptome, proteome, and regulatory RNA profiles with findings from genome-wide association studies.
SUMMARY: Studies employing these relatively new techniques promise to identify specific genes or regulatory RNAs with altered expression as beta-cell function begins to deteriorate in the spiral toward the development of diabetes. The ultimate goal is to identify the potential therapeutic targets to prevent beta-cell dysfunction and thereby better treat the individual with diabetes.
VIDEO ABSTRACT: http://links.lww.com/COE/A5.
Asparagine (N)-linked glycosylation is essential for efficient protein folding in the endoplasmic reticulum (ER) and anterograde trafficking through the secretory pathway. N-Glycans are attached to nascent polypeptides at consensus sites, N-X-T/S (X not equal P), by one of two enzymatic isoforms of the oligosaccharyltransferase (OST), STT3A or STT3B. Here, we examined the effect of the consensus site X and hydroxyl residue on the distributions of co- and post-translational N-glycosylation of a type I transmembrane glycopeptide scaffold. Using rapid radioactive pulse-chase experiments to resolve co-translational (STT3A) and post-translational (STT3B) events, we determined that NXS consensus sites containing large hydrophobic and negatively charged middle residues are frequently skipped by STT3A during protein translation. Post-translational modification of the cotranslationally skipped sites by STT3B was similarly hindered by the middle X residue, resulting in hypoglycosylation of NXS sites containing large hydrophobic and negatively charged side chains. In contrast, NXT consensus sites (barring NWT) were efficiently modified by the cotranslational machinery, reducing STT3B's role in modifying consensus sites skipped during protein translation. A strong correlation between cotranslational N-glycosylation efficiency and the rate of post-translational N-glycosylation was determined, showing that the OST STT3A and STT3B isoforms are similarly influenced by the hydroxyl and middle X consensus site residues. Substituting various middle X residues into an OST eubacterial homologous structure revealed that small and polar consensus site X residues fit well in the peptide binding site whereas large hydrophobic and negatively charged residues were harder to accommodate, indicating conserved enzymatic mechanisms for the mammalian OST isoforms.
Thousands of large intergenic noncoding RNAs (lincRNAs) have been identified in the mammalian genome, many of which have important roles in regulating a variety of biological processes. Here, we used a custom microarray to identify lincRNAs associated with activation of the innate immune response. A panel of 159 lincRNAs was found to be differentially expressed following innate activation of THP1 macrophages. Among them, linc1992 was shown to be expressed in many human tissues and was required for induction of TNFalpha expression. Linc1992 bound specifically to heterogenous nuclear ribonucleoprotein L (hnRNPL) and formed a functional linc1992-hnRNPL complex that regulated transcription of the TNFalpha gene by binding to its promoter. Transcriptome analysis revealed that linc1992 was required for expression of many immune-response genes, including other cytokines and transcriptional and posttranscriptional regulators of TNFalpha expression, and that knockdown of linc1992 caused dysregulation of these genes during innate activation of THP1 macrophages. Therefore, we named linc1992 THRIL (TNFalpha and hnRNPL related immunoregulatory LincRNA). Finally, THRIL expression was correlated with the severity of symptoms in patients with Kawasaki disease, an acute inflammatory disease of childhood. Collectively, our data provide evidence that lincRNAs and their binding proteins can regulate TNFalpha expression and may play important roles in the innate immune response and inflammatory diseases in humans.