A semi-centennial report on the participants depicted in Joel Babb's portrait, 'the first successful kidney transplantation'
Joseph Murray performed the first successful human kidney transplant on December 23, 1954. Forty-three years later, he along with participants Francis Moore and Leroy Vandam, commissioned a painting of the event from artist Joel Babb. To document this unique record of medical history, we identify all those present at the operation and depicted in the portrait, describe how the artist created the work, explain irregularities and inaccuracies in the painting, provide a 50-year follow-up on everyone involved, and comment on any influence this landmark event may have had on their subsequent careers.
Although some might argue about who is most deserving of credit for introducing anesthesia, there is no doubt that events of the 1840s marked the beginning of the conquest of pain through anesthesia. In 1882, at the urging of French portrait artist Carolus-Duran, and to fulfill training requirements as an art student in Paris, Robert Cutler Hinckley decided to create a painting of the first successful public demonstration of ether anesthesia. Hinckley's painting,The First Operation with Ether, has become one of the most popular paintings in medical history.
Before Hinckley began his work, sculptor John Quincy Adams Ward created the Ether Monument, which was erected and displayed in the Boston Public Garden in 1868. Several years after the celebration of the 150th anniversary of this historic event, the Board of Trustees at Massachusetts General Hospital, Boston, Massachusetts, at the urging of Dr. John B. Herman, commissioned Warren and Lucia Prosperi to create an accurate painting of the ether demonstration. The painting was unveiled on October 16, 2001, exactly 155 years after the original demonstration of ether anesthesia. It was presented as a gift to Massachusetts General Hospital by its physicians, nurses, and friends.
Although these paintings appear very similar, the documentation of a historic event by an artist, closer scrutiny reveals that the artists have used application of paint, control of light, varied points of view, and inclusion of detail to create works that differ markedly both in their appearance and in the emotional response they evoke.
The percutaneous approach for RF ablation of liver lesions high up in the dome is always difficult. The authors describe a transpulmonary approach that may be simpler and safer to use.
Deep sequencing can accurately measure the relative abundance of hundreds of mutations in a single bulk competition experiment, which can give a direct readout of the fitness of each mutant. Here we describe a protocol that we previously developed and optimized to measure the fitness effects of all possible individual codon substitutions for 10-aa regions of essential genes in yeast. Starting with a conditional strain (i.e., a temperature-sensitive strain), we describe how to efficiently generate plasmid libraries of point mutants that can then be transformed to generate libraries of yeast. The yeast libraries are competed under conditions that select for mutant function. Deep-sequencing analyses are used to determine the relative fitness of all mutants. This approach is faster and cheaper per mutant compared with analyzing individually isolated mutants. The protocol can be performed in ∼4 weeks and many 10-aa regions can be analyzed in parallel.
The genes of all organisms have been shaped by selective pressures. The relationship between gene sequence and fitness has tremendous implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Here, we have exploited deep sequencing technology to experimentally determine the fitness of all possible individual point mutants under controlled conditions for a nine-amino acid region of Hsp90. Over the past five decades, limited glimpses into the relationship between gene sequence and function have sparked a long debate regarding the distribution, relative proportion, and evolutionary significance of deleterious, neutral, and advantageous mutations. Our systematic experimental measurement of fitness effects of Hsp90 mutants in yeast, evaluated in the light of existing population genetic theory, are remarkably consistent with a nearly neutral model of molecular evolution.
In natural systems, selection acts on both protein sequence and expression level, but it is unclear how selection integrates over these two dimensions. We recently developed the EMPIRIC approach to systematically determine the fitness effects of all possible point mutants for important regions of essential genes in yeast. Here, we systematically investigated the fitness effects of point mutations in a putative substrate binding loop of yeast Hsp90 (Hsp82) over a broad range of expression strengths. Negative epistasis between reduced expression strength and amino acid substitutions was common, and the endogenous expression strength frequently obscured mutant defects. By analyzing fitness effects at varied expression strengths, we were able to uncover all mutant effects on function. The majority of mutants caused partial functional defects, consistent with this region of Hsp90 contributing to a mutation sensitive and critical process. These results demonstrate that important functional regions of proteins can tolerate mutational defects without experimentally observable impacts on fitness.
Evolution is the single cohesive logical framework in which all biological processes may exist simultaneously. Incremental changes in phenotype over imperceptibly large timescales have given rise to the enormous diversity of life we witness on earth both presently and through the natural record. The basic unit of evolution is mutation, and by perturbing biological processes, mutations may alter the fitness of an individual. However, the fitness effect of a mutation is difficult to infer from historical record, and complex to obtain experimentally in an efficient and accurate manner.
We have recently developed a high throughput method to iteratively mutagenize regions of essential genes in yeast and subsequently analyze individual mutant fitness termed Exceedingly Methodical and Parallel Investigation of Randomized Individual Codons (EMPIRIC). Utilizing this technique as exemplified in Chapters II and III, it is possible to determine the fitness effects of all possible point mutations in parallel through growth competition followed by a high throughput sequencing readout. We have employed this technique to determine the distribution of fitness effects in a nine amino acid region of the Hsp90 gene of S. cerevisiae under elevated temperature, and found the bimodal distribution of fitness effects to be remarkably consistent with near-neutral theory. Comparing the measured fitness effects of mutants to the natural record, phylogenetic alignments appear to be a poor predictor of experimental fitness.
In Chapter IV, to further interrogate the properties of this region, library competition under conditions of elevated temperature and salinity were performed to study the potential of protein adaptation. Strikingly, whereas both optimal and elevated temperatures produced no statistically significant beneficial mutations, under conditions of elevated salinity, adaptive mutations appear with fitness advantages up to 8% greater than wild type. Of particular interest, mutations conferring fitness benefits under conditions of elevated salinity almost always experience a fitness defect in other experimental conditions, indicating these mutations are environmentally specialized. Applying the experimental fitness measurements to long standing theoretical predictions of adaptation, our results are remarkably consistent with Fisher’s Geometric Model of protein evolution.
Epistasis between mutations can have profound effects on evolutionary trajectories. Although the importance of epistasis has been realized since the early 1900s, the interdependence of mutations is difficult to study in vivo due to the stochastic and constant nature of background mutations. In Chapter V, utilizing the EMPIRIC methodology allows us to study the distribution of fitness effects in the context of mutant genetic backgrounds with minimal influence from unintended background mutations. By analyzing intragenic epistatic interactions, we uncovered a complex interplay between solvent shielded structural residues and solvent exposed hydrophobic surface in the amino acid 582-590 region of Hsp90. Additionally, negative epistasis appears to be negatively correlated with mutational promiscuity while additive interactions are positively correlated, indicating potential avenues for proteins to navigate fitness ‘valleys’.
In summary, the work presented in this dissertation is focused on applying experimental context to the theory-rich field of evolutionary biology. The development and implementation of a novel methodology for the rapid and accurate assessment of organismal fitness has allowed us to address some of the most basic processes of evolution including adaptation and protein expression level. Through the work presented here and by investigators across the world, the application of experimental data to evolutionary theory has the potential to improve drug design and human health in general, as well as allow for predictive medicine in the coming era of personalized medicine.
Adjuvant-Specific Serum Cytokine Profiles in the Context of a DNA Prime-Protein Boost HIV-1 Vaccine: A Dissertation
In recent years, heterologous prime-boost vaccination constructs have emerged as a promising strategy to generate broad and protective immunity against a variety of pathogens. The utility of DNA vaccination in priming the immune system, in particular, has improved the immunogenicity of vaccines against difficult pathogens such as HIV-1. In addition, many vaccine formulations include an adjuvant to augment immune responses. However, the mechanisms and profiles of many adjuvants remain largely unknown, particularly in the context of such combination immunization approaches.
My thesis research studied the effects of several adjuvants, QS-21, aluminum hydroxide, MPL, and ISCOMATRIX™ adjuvant in the context of a previously described pentavalent HIV-1 Env DNA prime-protein boost vaccine, DP6-001. In a murine model, we quantified HIV antigen-specific humoral and T cell responses, as well as pro-inflammatory serum cytokine and chemokines, both shortly after immunization and at the termination of studies. Our data indicates that each candidate adjuvant generates a unique pattern of biomarkers as well as improved immunogenicity in the context of the DP6-001 DNA prime-protein boost vaccine.
Additionally, we examined the impact of several innate signaling pathways on the adaptive immunity raised by DP6-001 and adjuvants, as well as on the unique serum cytokine profiles. These studies provide valuable information in selection of an adjuvant for inclusion in future prime-boost strategies, with the goal of enhancing immunogenicity while minimizing reactogenicity. Furthermore, these studies provided insight about the utility of different current adjuvants in a prime-boost formulation, and the unique immune environment induced by DNA priming.
Transcriptional and Translational Mechanisms Controlling Circadian Rhythms in Drosophila: A Dissertation
Circadian rhythms are self-sustained 24-hour period oscillations present in most organisms, from bacteria to human. They can be synchronized to external cues, thus allowing organisms to anticipate environmental variations and optimize their performance in nature.
In Drosophila, the molecular pacemaker consists of two interlocked transcriptional feedback loops. CLOCK/CYCLE (CLK/CYC) sits in the center and drives rhythmic transcription of period (per), timeless (tim), vrille (vri) and PAR domain protein 1 (Pdp1). PER and TIM negatively feedback on CLK/CYC transcriptional activity, forming one loop, while VRI and PDP1 form the other by regulating Clk transcription negatively and positively, respectively. Posttranscriptional and posttranslational regulations also contribute to circadian rhythms. Although much has been learned about these feedback loops, we are still far from understanding how stable 24-hour period rhythms are generated.
My thesis work was to determine by which molecular mechanisms kayak-α (kay-α) and Ataxin-2 (Atx2) regulate Drosophila circadian behavior. Both genes are required for the precision of circadian rhythms since knocking down either gene in circadian pacemaker neurons results in long period phenotype.
The work on kay-α constitutes the first half of my thesis. We found that the transcription factor KAY-α can bind to VRI and inhibit VRI’s repression on the Clk promoter. Interestingly, KAY-α can also repress CLK’s transcriptional activity on its target genes (e.g., per and tim). Therefore, KAY-α is proposed to bring precision and stability to the molecular pacemaker by regulating both transcriptional loops.
The second half of my thesis focuses on ATX2, an RNA binding protein whose mammalian homolog has been implicated in neurodegenerative diseases. We found that ATX2 is required for PER accumulation in circadian pacemaker neurons. It forms a complex with TWENTY-FOUR (TYF)—a crucial activator of PER translation—and promotes TYF’s interaction with Poly(A)-binding protein. This work reveals the role of ATX2 in the control of circadian rhythms as an activator of PER translation, in contrast to its well-established role as a repressor of translation. It also further demonstrates the importance of translational regulation on circadian rhythms. Finally, it may help understanding how ATX2 causes neuronal degeneration in human diseases.
Surgery volume, quality of care and operative mortality in coronary artery bypass graft surgery: a re-examination using fixed-effects regression
For many surgical procedures, apparent volume–outcome relationships may reflect differences in patient risk-profiles as well as quality of care. As some important patient profile differences may be unobserved, we use fixed effects (FE) regression to estimate the relationship between operative mortality and surgeon and hospital volumes, and compare this method with the more commonly used random effects (RE) regression approach. The 1998 and 1999 Medicare Inpatient and Denominator files for Medicare Fee for Service enrollees aged 65–99. Operative mortality rates are estimated for different surgeon and hospital volume tertiles (high, medium, low) using FE and RE regression methods, adjusted for patient demographics and morbidities. The data were collected by the Centers for Medicare and Medicaid Services (CMS). FE regression estimates that lowest volume tertile hospitals have 1.4 and lowest volume tertile surgeons have 1.6 additional operative deaths (for every 100 CABG surgeries) compared to their highest volume tertile counterparts. The corresponding RE estimates are 0.5 and 1.4 respectively. The substantially higher FE hospital volume effect compared to RE indicates the presence of unobserved “protective” characteristics in lower volume providers, including a less complicated patient profile. Lower hospital and surgeon volumes are associated with substantially higher excess operative mortality from CABG surgeries than previously estimated.
Risk adjustment models can establish appropriate payments and incentives for delivering superior primary care, particularly to people with chronic conditions, through health-based capitation and performance assessment in a patient-centered medical home (PCMH). The practical issues and administrative structures for implementing bundled PCMH payment that we discuss are relevant for single-payer Scandinavian countries as well as the US. Feasibility is supported by the “virtual all-payer” PCMH pilot of one US health plan.
Recent years have seen a wave of laws making it more difficult to vote in the United States. Their ostensible purpose is to prevent voting by persons not legally qualified, and thus to improve public confidence in electoral integrity. Are such laws in fact needed to address serious fraud problems? On the other hand, how many and what kinds of legitimate voters will be, or have been, disenfranchised by them? Is voter confidence positively, or negatively, affected by voter ID laws? This article surveys what is known about these issues and offers suggestions for how statisticians can contribute.
From the Preface: The Centers for Medicare and Medicaid Services (CMS), through a subcontract with Yale New Haven Health Services Corporation, Center for Outcomes Research and Evaluation (YNHHSC/CORE), is supporting a committee appointed by the Committee of Presidents of Statistical Societies (COPSS) to address statistical issues identified by the CMS and stakeholders about CMS’s approach to modeling hospital quality based on outcomes. In the spring of 2011, with the direct support of YNHHSC/ CORE, COPSS formed a committee comprised of one member from each of its constituent societies, a chair, and a staff member from the American Statistical Association, and held a preliminary meeting in April. In June, YNHHSC/CORE executed a subcontract with COPSS under its CMS contract to support the development of a White Paper on statistical modeling. Specifically, YNHHSC/CORE contracted with COPSS to “provide guidance on statistical approaches . . .when estimating performance metrics,” and “consider and discuss concerns commonly raised by stakeholders (hospitals, consumer, and insurers) about the use of “hierarchical generalized linear models in profiling hospital quality. The committee convened in June and August of 2011, and exchanged a wide variety of materials. To ensure the committee’s independence, YNHHSC/CORE did not comment on the white paper findings, and CMS pre-cleared COPSS’ publication of an academic manuscript based on the White Paper.
INR targets and site-level anticoagulation control: results from the Veterans AffaiRs Study to Improve Anticoagulation (VARIA)
BACKGROUND: Not all clinicians target the same International Normalized Ratio (INR) for patients with a guideline-recommended target range of 2-3. A patient's mean INR value suggests the INR that was actually targeted. We hypothesized that sites would vary by mean INR, and that sites of care with mean values nearest to 2.5 would achieve better anticoagulation control, as measured by per cent time in therapeutic range (TTR).
OBJECTIVES: To examine variations among sites in mean INR and the relationship with anticoagulation control in an integrated system of care.
PATIENTS/METHODS: We studied 103,897 patients receiving oral anticoagulation with an expected INR target between 2 and 3 at 100 Veterans Health Administration (VA) sites from 1 October 2006 to 30 September 2008. Key site-level variables were: proportion near 2.5 (that is, percentage of patients with mean INR between 2.3 and 2.7) and mean risk-adjusted TTR.
RESULTS: Site mean INR ranged from 2.22 to 2.89; proportion near 2.5, from 30 to 64%. Sites' proportions of patients near 2.5, below 2.3 and above 2.7 were consistent from year to year. A 10 percentage point increase in the proportion near 2.5 predicted a 3.8 percentage point increase in risk-adjusted TTR (P < 0.001).
CONCLUSIONS: Proportion of patients with mean INR near 2.5 is a site-level 'signature' of care and an implicit measure of targeted INR. This proportion varies by site and is strongly associated with site-level TTR. Our study suggests that sites wishing to improve TTR, and thereby improve patient outcomes, should avoid the explicit or implicit pursuit of non-standard INR targets.
BACKGROUND: Warfarin is effective in preventing thromboembolic events, but concerns exist regarding its use in patients with substance abuse.
OBJECTIVE: Identify which patients with substance abuse who receive warfarin are at risk for poor outcomes.
DESIGN: Retrospective cohort study. Diagnostic codes, lab values, and other factors were examined to identify risk of adverse outcomes.
PATIENTS: Veterans AffaiRs Study to Improve Anticoagulation (VARIA) database of 103,897 patients receiving warfarin across 100 sites.
MAIN MEASURES: Outcomes included percent time in therapeutic range (TTR), a measure of anticoagulation control, and major hemorrhagic events by ICD-9 codes.
RESULTS: Nonusers had a higher mean TTR (62 %) than those abusing alcohol (53 %), drugs (50 %), or both (44 %, p < 0.001). Among alcohol abusers, an increasing ratio of the serum hepatic transaminases aspartate aminotransferase/alanine aminotransferase (AST:ALT) correlated with inferior anticoagulation control; normal AST:ALT ≤ 1.5 predicted relatively modest decline in TTR (54 %, p < 0.001), while elevated ratios (AST:ALT 1.50-2.0 and > 2.0) predicted progressively poorer anticoagulation control (49 % and 44 %, p < 0.001 compared to nonusers). Age-adjusted hazard ratio for major hemorrhage was 1.93 in drug and 1.37 in alcohol abuse (p < 0.001 compared to nonusers), and remained significant after also controlling for anticoagulation control and other bleeding risk factors (1.69 p < 0.001 and 1.22 p = 0.003). Among alcohol abusers, elevated AST:ALT >2.0 corresponded to more than three times the hemorrhages (HR 3.02, p < 0.001 compared to nonusers), while a normal ratio AST:ALT ≤ 1.5 predicted a rate similar to nonusers (HR 1.19, p < 0.05).
CONCLUSIONS: Anticoagulation control is particularly poor in patients with substance abuse. Major hemorrhages are more common in both alcohol and drug users. Among alcohol abusers, the ratio of AST/ALT holds promise for identifying those at highest risk for adverse events.
Using the bootstrap to establish statistical significance for relative validity comparisons among patient-reported outcome measures
BACKGROUND: Relative validity (RV), a ratio of ANOVA F-statistics, is often used to compare the validity of patient-reported outcome (PRO) measures. We used the bootstrap to establish the statistical significance of the RV and to identify key factors affecting its significance.
METHODS: Based on responses from 453 chronic kidney disease (CKD) patients to 16 CKD-specific and generic PRO measures, RVs were computed to determine how well each measure discriminated across clinically-defined groups of patients compared to the most discriminating (reference) measure. Statistical significance of RV was quantified by the 95% bootstrap confidence interval. Simulations examined the effects of sample size, denominator F-statistic, correlation between comparator and reference measures, and number of bootstrap replicates.
RESULTS: The statistical significance of the RV increased as the magnitude of denominator F-statistic increased or as the correlation between comparator and reference measures increased. A denominator F-statistic of 57 conveyed sufficient power (80%) to detect an RV of 0.6 for two measures correlated at r = 0.7. Larger denominator F-statistics or higher correlations provided greater power. Larger sample size with a fixed denominator F-statistic or more bootstrap replicates (beyond 500) had minimal impact.
CONCLUSIONS: The bootstrap is valuable for establishing the statistical significance of RV estimates. A reasonably large denominator F-statistic (F > 57) is required for adequate power when using the RV to compare the validity of measures with small or moderate correlations (r < 0.7). Substantially greater power can be achieved when comparing measures of a very high correlation (r > 0.9).
The factors that limit primary care providers (PCPs) from intervening for adults with evolving acute severe illness are less understood than the increasing frequency of management by acute care providers.
Rates of pre-hospital patient management by a PCP and of communication with the acute care teams were measured in a multi-center cross sectional descriptive study conducted in all four of the adult medical intensive care units (ICU) of the 3 hospitals in central Massachusetts that provide tertiary care. Rates were measured for 390 critical care encounters using a validated instrument to abstract the medical record and conduct telephone interviews.
PCPs implemented pre-hospital management for 8 episodes of acute illness among 300 encounters. Infrequent pre-hospital management by PCPs was attributed to their lack of awareness of the patient's evolving acute illness. Only 21% of PCPs were aware of the acute illness before their patient was admitted to an ICU and 33% were not aware that their patient was in an ICU. Rates of PCP involvement were not appreciably different among provider groups, by patient age, sex, insurance status, hospital, ICU, or ICU staffing model.
We identified lack of PCP awareness of the acute illness and high rates of PCP referral to acute care providers as the most frequent barriers to pre-hospital management of evolving acute illness. These findings suggest that implementing processes that encourage early patient-PCP communication and increase rates of prehospital management of infections and acute exacerbations of chronic diseases could reduce utilization of acute care services.
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Tumor dissemination to the extra-hepatic region of the portal vein, lymph nodes, lungs or bones contributes to the high mortality seen in HCC; yet, the molecular mechanisms responsible for HCC metastasis remain unclear. Prior studies have suggested a potential link between accumulated cytoplasm-localized p16 and tumor progression. Here we report that p16 enhances metastasis-associated phenotypes in HCC cells - ectopic p16 expression increased cell migration in vitro, and lung colonization after intravenous injection, whereas knockdown of endogenous p16 reduced cell migration. Interestingly, analysis of p16 mutants indicated that the Cdk4 interaction domain is required for stimulation of HCC cell migration; however, knockdown of Cdk4 and Cdk6 showed that these proteins are dispensable for this phenomenon. Intriguingly, we found that in p16-positive HCC samples, p16 protein is predominantly localized in the cytoplasm. In addition, we identified a potential role for nuclear-cytoplasmic shuttling in p16-stimulated migration, consistent with the predominantly cytoplasmic localization of p16 in IHC-positive HCC samples. Finally, we determined that p16-stimulated cell migration requires the Cdc42 GTPase. Our results demonstrate for the first time a pro-migratory role for p16, and suggest a potential mechanism for the observed association between cytoplasmic p16 and tumor progression in diverse tumor types.
The study of human evolution is of interest to many both for the potential it has to improve our understanding of heritable disease, as well as for the possibility of illuminating evidence for adaptations that may help to tell the story of our origin. But uncovering evidence of positive selection at the genetic level has been a challenge. It remains unclear how much of the human genome has been affected by positive selection, what the main mechanism of selection is, and what types of patterns we should be looking for to identify adaptations. With whole-genome sequencing and high performance computation, we are quickly shifting to a field in which data is no longer a limiting factor. Here we will discuss the progress that has been made towards these ends, explore the best examples of human-specific adaptations to date, and discuss the implications of these findings within the context of classical population genetic theory.
Survival response to increased ceramide involves metabolic adaptation through novel regulators of glycolysis and lipolysis
The sphingolipid ceramide elicits several stress responses, however, organisms survive despite increased ceramide but how they do so is poorly understood. We demonstrate here that the AKT/FOXO pathway regulates survival in increased ceramide environment by metabolic adaptation involving changes in glycolysis and lipolysis through novel downstream targets. We show that ceramide kinase mutants accumulate ceramide and this leads to reduction in energy levels due to compromised oxidative phosphorylation. Mutants show increased activation of Akt and a consequent decrease in FOXO levels. These changes lead to enhanced glycolysis by upregulating the activity of phosphoglyceromutase, enolase, pyruvate kinase, and lactate dehydrogenase to provide energy. A second major consequence of AKT/FOXO reprogramming in the mutants is the increased mobilization of lipid from the gut through novel lipase targets, CG8093 and CG6277 for energy contribution. Ubiquitous reduction of these targets by knockdown experiments results in semi or total lethality of the mutants, demonstrating the importance of activating them. The efficiency of these adaptive mechanisms decreases with age and leads to reduction in adult life span of the mutants. In particular, mutants develop cardiac dysfunction with age, likely reflecting the high energy requirement of a well-functioning heart. The lipases also regulate physiological triacylglycerol homeostasis and are important for energy metabolism since midgut specific reduction of them in wild type flies results in increased sensitivity to starvation and accumulation of triglycerides leading to cardiac defects. The central findings of increased AKT activation, decreased FOXO level and activation of phosphoglyceromutase and pyruvate kinase are also observed in mice heterozygous for ceramide transfer protein suggesting a conserved role of this pathway in mammals. These data reveal novel glycolytic and non-autonomous lipolytic pathways in response to increased ceramide for sustenance of high energy demanding organ functions like the heart.