Early events in folding can determine if a protein is going to fold, misfold, or aggregate. Understanding these deterministic events is paramount for de novo protein engineering, the enhancement of biopharmaceutical stabilities, and understanding neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. However, the physicochemical and structural biases within high energy states of protein biopolymers are poorly understood.
A combined experimental and computational study was conducted on the small β/α-repeat protein CheY to determine the structural basis of its submillisecond misfolding reaction to an off-pathway intermediate. Using permutations, we were able to discriminate between the roles of two proposed mechanisms of folding; a nucleation condensation model, and a hydrophobic collapse model driven by the formation of clusters of isoleucine, leucine, and valine (ILV) residues. We found that by altering the ILV cluster connectivity we could bias the early folding events to either favor on or off-pathway intermediates.
Structural biases were also experimentally observed in the unfolded state of a de novo designed synthetic β/α-repeat protein, Di-III_14. Although thermodynamically and kinetically 2-state, Di-III_14 has a well structured unfolded state that is only observable under native-favoring conditions. This unfolded state appears to retain native-like structure, consisting of a hydrophobic 7 core (69% ILV) stabilized by solvent exposed polar groups and long range electrostatic interactions.
Together, these results suggest that early folding events are largely deterministic in these two systems. Generally, low contact order ILV clusters favor local compaction and, in specific cases, long range electrostatic interactions may have stabilizing effects in higher energy states.
Long-Term Survival and Prognostic Factors in Patients with Acute Decompensated Heart Failure According to Ejection Fraction Findings: A Population-Based Perspective: A Master Thesis
Limited data exists describing the long-term prognosis of patients with acute decompensated heart failure (ADHF) further stratified according to currently recommended ejection fraction (EF) findings. In addition, little is known about the magnitude of, and factors associated with, long-term prognosis for these patients. Based on previously validated and clinically relevant criteria, we defined HF-REF as patients with an EF value ≤40%, HF-PEF was defined as an EF value > 50%, and HF-BREF was defined as patients with an EF value during their index hospitalization between 41 and 49%. The hospital medical records of residents of the Worcester (MA) metropolitan area who were discharged after ADHF from all 11 medical centers in central Massachusetts during the 5 study years of 1995, 2000, 2002, 2004, and 2006 were reviewed. Follow-up was completed through 2011 for all patient cohorts. The average age of this population was 75 years, the majority was white, and 44% were men. Patients with HF-PEF experienced higher post discharge survival rates than patients with either HF-REF or HF-BREF at 1, 2, and 5-years after discharge. Advanced age and lower estimated glomerular filtration rate findings at the time of hospital admission were important predictors of 1-year death rates, irrespective of EF findings. Previously diagnosed chronic obstructive pulmonary disease, chronic kidney disease, and atrial fibrillation were associated with a poor prognosis in patients with PEF and REF whereas a history of diabetes was an important prognostic factor for patients with REF and BREF. In conclusion, although improvements in 1-year post-discharge survival were observed for patients in each of the 3 EF groups examined to varying degrees, the post- 7 discharge prognosis of all patients with ADHF remains guarded. In addition, we observed differences in several prognostic factors between patients with ADHF with varying EF findings, which have implications for more refined treatment and surveillance plans for these patients.
Short noncoding RNAs play roles in regulating nearly every biological process, in nearly every organism, yet the exact function and importance of these molecules remains a subject of some debate. In order to gain a better understanding of the contexts in which these regulators have evolved, I have undertaken a variety of approaches to study the evolutionary history of the components that make up these pathways, in the form of two main research efforts. In the first chapter, I have used a combination of population genetics and molecular evolution techniques to show that proteins involved in the piRNA pathway are rapidly evolving, and that different components of the pathway seem to be evolving rapidly on different timescales. These rapidly evolving piRNA pathway proteins can be loosely separated into two groups. The first group appears to evolve quickly at the species level, perhaps in response to transposons that invade across species lines, while the second group appears to evolve quickly at the level of individual populations, perhaps in response to transposons that are paternally present yet novel to the maternal genome. In the second chapter of my research, I have used molecular evolution techniques and carefully devised controls to show that the binding sites of well-conserved miRNAs are among the most slowly changing short motifs in the genome, consistent with a conserved function for these short RNAs in regulatory pathways that are ancient and extremely slow to change. I have additionally discovered a major flaw in an existing approach to motif turnover calculations, which may lead to systematic biases in the published literature toward the false inference of increased regulatory complexity over time. I have implemented a revised approach to motif turnover that addresses this flaw.
Background: The initial systolic blood pressure (SBP) in patients with acute heart failure (AHF) can be used as a guide when choosing specific pharmacologic treatments by helping identify the underlying type of HF (e.g., HF with preserved ejection fraction). Clinical experience and research data from our medical center suggests that AHF with elevated SBP may be presenting less frequently than in the past. This may call into question the utility of initial SBP as a clinical guide. The goal of this Master’s Thesis is to test the hypothesis that the frequency of AHF patients with a SBP>160mmhg has declined over time.
Methods: This observational study compares data from 4 cohorts of adult patients admitted with AHF in central MA. Data were obtained from a contemporary (2011-2013) study of patients with AHF as well as from the 1995, 2000, 2006 Worcester Heart Failure Study (WHFS) cohorts. The Framingham criteria the diagnostic criterion for AHF. The main outcome was the proportion of patients with AHF with a SBP > 160 mmHg who presented in each of the 4 study cohorts and was examined by multivariate logistic regression.
Results: 2,366 patients comprised the study population. The average age was 77 years, 55% were female, 94% white, and 75% had prior HF. In 1995 33.6% of AHF patients had a SBP >160 mmHg compared to 19.5% in 2011-2013 (p160 mmHg in 2006 (0.64, (0.42-0.96)) and 2011-13 (0.46, (0.28-0.74)).
Conclusion: The proportion of patients with AHF and an initial SBP >160 mmHg has significantly declined over time. This may warrant a reexamination of the utility of SBP to inform diagnosis and treatment in patients with AHF.
The characteristics of nurses acting as organizational champions, as well as the ways that clinical leaders systematically harness the energy of these champions in support of innovation, were explored in this qualitative descriptive study. The specific aims were guided by prior empirical evidence and identified research needs. Semi-structured interviews were conducted with 14 formal nursing leaders (e.g. managers, educators, administration) in an academic medical center. This study, including the interview guide, was informed by Kouzes and Posner’s (2007) Five Practices of Exemplary Leadership. Two models were developed to describe the data. Overall, participants echoed prior empirical findings identifying a need for organizational champions’ support of innovation and explained how some nurses seem to have “innate” characteristics that make them champions. Participants identified the champion as the “go to” person who can see the bigger picture and who seems to “own their own practice”. They described the importance of being truly present on the unit in order to harness the energy of these champions. Once champions are identified, leaders match the champions’ talents to the innovation planned, secure buy in from the champions, and actively work to support champions and get a culture of innovation “in the drinking water.” This work enhances the leader’s experience and makes him/her feel inspired and engaged. The two models developed based on the participants’ description of their experience working with staff nurses acting as organizational champions provide a framework for clinical leaders to identify and engage organizational champions in their clinical areas in support of innovation.
Comment on: Transcoronary concentration gradients of circulating microRNAs. [Circulation. 2011]
The Drosophila let-7-Complex (let-7-C) is a polycistronic locus encoding three ancient microRNAs: let-7, miR-100, and fly lin-4 (miR-125). We find that the let-7-C locus is principally expressed in the pupal and adult neuromusculature. let-7-C knockout flies appear normal externally but display defects in adult behaviors (e.g., flight, motility, and fertility) as well as clear juvenile features in their neuromusculature. We find that the function of let-7-C to ensure the appropriate remodeling of the abdominal neuromusculature during the larval-to-adult transition is carried out predominantly by let-7 alone. This heterochronic role of let-7 is likely just one of the ways in which let-7-C promotes adult behavior.
mirWIP: microRNA target prediction based on microRNA-containing ribonucleoprotein-enriched transcripts
Target prediction for animal microRNAs (miRNAs) has been hindered by the small number of verified targets available to evaluate the accuracy of predicted miRNA-target interactions. Recently, a dataset of 3,404 miRNA-associated mRNA transcripts was identified by immunoprecipitation of the RNA-induced silencing complex components AIN-1 and AIN-2. Our analysis of this AIN-IP dataset revealed enrichment for defining characteristics of functional miRNA-target interactions, including structural accessibility of target sequences, total free energy of miRNA-target hybridization and topology of base-pairing to the 5' seed region of the miRNA. We used these enriched characteristics as the basis for a quantitative miRNA target prediction method, miRNA targets by weighting immunoprecipitation-enriched parameters (mirWIP), which optimizes sensitivity to verified miRNA-target interactions and specificity to the AIN-IP dataset. MirWIP can be used to capture all known conserved miRNA-mRNA target relationships in Caenorhabditis elegans at a lower false-positive rate than can the current standard methods.
Our appreciation of the significance of microRNAs to biology at large continues to be an evolving process.
microRNAs comprise a few percent of animal genes and have been recognized as important regulators of a diverse range of biological processes. Understanding the biological functions of miRNAs requires effective means to identify their targets. Combined efforts from computational prediction, miRNA over-expression or depletion, and biochemical purification have identified thousands of potential miRNA-target pairs in cells and organisms. Complementarity to the miRNA seed sequence appears to be a common principle in target recognition. Other features, including miRNA-target duplex stability, binding site accessibility, and local UTR structure might affect target recognition. Yet computational approaches using such contextual features have yielded largely nonoverlapping results and experimental assessment of their impact has been limited. Here, we compare two large sets of miRNA targets: targets identified using an improved Ago1 immunopurification method and targets identified among transcripts up-regulated after Ago1 depletion. We found surprisingly limited overlap between these sets. The two sets showed enrichment for target sites with different molecular, structural and functional properties. Intriguingly, we found a strong correlation between UTR length and other contextual features that distinguish the two groups. This finding was extended to all predicted microRNA targets. Distinct repression mechanisms could have evolved to regulate targets with different contextual features. This study reveals a complex relationship among different features in miRNA-target recognition and poses a new challenge for computational prediction.
Comment on: The cyclin-dependent kinase inhibitors, cki-1 and cki-2, act in overlapping but distinct pathways to control cell-cycle quiescence during C. elegans development. Buck SH, et al. Cell Cycle 2009; 8:2613-20.
A feedback circuit involving let-7-family miRNAs and DAF-12 integrates environmental signals and developmental timing in Caenorhabditis elegans
Animal development is remarkably robust; cell fates are specified with spatial and temporal precision despite physiological and environmental contingencies. Favorable conditions cause Caenorhabditis elegans to develop rapidly through four larval stages (L1-L4) to the reproductive adult. In unfavorable conditions, L2 larvae can enter the developmentally quiescent, stress-resistant dauer larva stage, enabling them to survive for prolonged periods before completing development. A specific progression of cell division and differentiation events occurs with fidelity during the larval stages, regardless of whether an animal undergoes continuous or dauer-interrupted development. The temporal patterning of developmental events is controlled by the heterochronic genes, whose products include microRNAs (miRNAs) and regulatory proteins. One of these proteins, the DAF-12 nuclear hormone receptor, modulates the transcription of certain let-7-family miRNAs, and also mediates the choice between the continuous vs. dauer-interrupted life history. Here, we report a complex feedback loop between DAF-12 and the let-7-family miRNAs involving both the repression of DAF-12 by let-7-family miRNAs and the ligand-modulated transcriptional activation and repression of the let-7-Fam miRNAs by DAF-12. We propose that this feedback loop functions to ensure robustness of cell fate decisions and to coordinate cell fate with developmental arrest.
Why do many microRNA gene mutants display no evident phenotype? Multiply mutant worms that are selectively impaired in genetic regulatory network activities have been used to uncover previously unknown functions for numerous Caenorhabditis elegans microRNAs.
The developmental timing regulator HBL-1 modulates the dauer formation decision in Caenorhabditis elegans
Animals developing in the wild encounter a range of environmental conditions, and so developmental mechanisms have evolved that can accommodate different environmental contingencies. Harsh environmental conditions cause Caenorhabditis elegans larvae to arrest as stress-resistant "dauer" larvae after the second larval stage (L2), thereby indefinitely postponing L3 cell fates. HBL-1 is a key transcriptional regulator of L2 vs. L3 cell fate. Through the analysis of genetic interactions between mutations of hbl-1 and of genes encoding regulators of dauer larva formation, we find that hbl-1 can also modulate the dauer formation decision in a complex manner. We propose that dynamic interactions between genes that regulate stage-specific cell fate decisions and those that regulate dauer formation promote the robustness of developmental outcomes to changing environmental conditions.
Animals have evolved mechanisms to ensure the robustness of developmental outcomes to changing environments. MicroRNA expression may contribute to developmental robustness because microRNAs are key post-transcriptional regulators of developmental gene expression and can affect the expression of multiple target genes. Caenorhabditis elegans provides an excellent model to study developmental responses to environmental conditions. In favorable environments, C. elegans larvae develop rapidly and continuously through four larval stages. In contrast, in unfavorable conditions, larval development may be interrupted at either of two diapause stages: The L1 diapause occurs when embryos hatch in the absence of food, and the dauer diapause occurs after the second larval stage in response to environmental stimuli encountered during the first two larval stages. Dauer larvae are stress resistant and long lived, permitting survival in harsh conditions. When environmental conditions improve, dauer larvae re-enter development, and progress through two post-dauer larval stages to adulthood. Strikingly, all of these life history options (whether continuous or interrupted) involve an identical pattern and sequence of cell division and cell fates. To identify microRNAs with potential functions in buffering development in the context of C. elegans life history options, we used multiplex real-time PCR to assess the expression of 107 microRNAs throughout development in both continuous and interrupted life histories. We identified 17 microRNAs whose developmental profile of expression is affected by dauer life history and/or L1 diapause, compared to continuous development. Hence these microRNAs could function to regulate gene expression programs appropriate for different life history options in the developing worm.
MicroRNAs regulate temporal transitions in gene expression associated with cell fate progression and differentiation throughout animal development. Genetic analysis of developmental timing in the nematode Caenorhabditis elegans identified two evolutionarily conserved microRNAs, lin-4/mir-125 and let-7, that regulate cell fate progression and differentiation in C. elegans cell lineages. MicroRNAs perform analogous developmental timing functions in other animals, including mammals. By regulating cell fate choices and transitions between pluripotency and differentiation, microRNAs help to orchestrate developmental events throughout the developing animal, and to play tissue homeostasis roles important for disease, including cancer.
Dauer larva quiescence alters the circuitry of microRNA pathways regulating cell fate progression in C. elegans
In C. elegans larvae, the execution of stage-specific developmental events is controlled by heterochronic genes, which include those encoding a set of transcription factors and the microRNAs that regulate the timing of their expression. Under adverse environmental conditions, developing larvae enter a stress-resistant, quiescent stage called 'dauer'. Dauer larvae are characterized by the arrest of all progenitor cell lineages at a stage equivalent to the end of the second larval stage (L2). If dauer larvae encounter conditions favorable for resumption of reproductive growth, they recover and complete development normally, indicating that post-dauer larvae possess mechanisms to accommodate an indefinite period of interrupted development. For cells to progress to L3 cell fate, the transcription factor Hunchback-like-1 (HBL-1) must be downregulated. Here, we describe a quiescence-induced shift in the repertoire of microRNAs that regulate HBL-1. During continuous development, HBL-1 downregulation (and consequent cell fate progression) relies chiefly on three let-7 family microRNAs, whereas after quiescence, HBL-1 is downregulated primarily by the lin-4 microRNA in combination with an altered set of let-7 family microRNAs. We propose that this shift in microRNA regulation of HBL-1 expression involves an enhancement of the activity of lin-4 and let-7 microRNAs by miRISC modulatory proteins, including NHL-2 and LIN-46. These results illustrate how the employment of alternative genetic regulatory pathways can provide for the robust progression of progenitor cell fates in the face of temporary developmental quiescence.
Like mammalian neurons, Caenorhabditis elegans neurons lose axon regeneration ability as they age, but it is not known why. Here, we report that let-7 contributes to a developmental decline in anterior ventral microtubule (AVM) axon regeneration. In older AVM axons, let-7 inhibits regeneration by down-regulating LIN-41, an important AVM axon regeneration-promoting factor. Whereas let-7 inhibits lin-41 expression in older neurons through the lin-41 3' untranslated region, lin-41 inhibits let-7 expression in younger neurons through Argonaute ALG-1. This reciprocal inhibition ensures that axon regeneration is inhibited only in older neurons. These findings show that a let-7-lin-41 regulatory circuit, which was previously shown to control timing of events in mitotic stem cell lineages, is reutilized in postmitotic neurons to control postdifferentiation events.
In metazoans, microRNAs play a critical role in the posttranscriptional regulation of genes required for cell proliferation and differentiation. MicroRNAs themselves are regulated by a multitude of mechanisms influencing their transcription and posttranscriptional maturation. However, there is only sparse knowledge on pathways regulating the mature, functional form of microRNA. Here, we uncover the implication of the decapping scavenger protein DCS-1 in the control of microRNA turnover. In Caenorhabditis elegans, mutations in dcs-1 increase the levels of functional microRNAs. We demonstrate that DCS-1 interacts with the exonuclease XRN-1 to promote microRNA degradation in an independent manner from its known decapping scavenger activity, establishing two molecular functions for DCS-1. Our findings thus indicate that DCS-1 is part of a degradation complex that performs microRNA turnover in animals.
Interview with Victor Ambros, who studies how microRNAs impact development.