Nicholas Mitsakakis

Exploration of Essential Gene Functions via Titratable Promoter Alleles

Nearly 20% of yeast genes are required for viability, hindering genetic analysis with knockouts. We created promoter-shutoff strains for over two-thirds of all essential yeast genes and subjected them to morphological analysis, size profiling, drug sensitivity screening, and microarray expression profiling. We then used this compendium of data to ask which phenotypic features characterized different functional classes and used these to infer potential functions for uncharacterized genes. We identified genes involved in ribosome biogenesis (HAS1, URB1, and URB2), protein secretion (SEC39), mitochondrial import (MIM1), and tRNA charging (GSN1). In addition, apparent negative feedback transcriptional regulation of both ribosome biogenesis and the proteasome was observed. We furthermore show that these strains are compatible with automated genetic analysis. This study underscores the importance of analyzing mutant phenotypes and provides a resource to complement the yeast knockout collection.

A Panoramic View of Yeast Noncoding RNA Processing

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.

Ultrasound assessment of gastric content and volume.

Background:Aspiration of gastric contents can be a serious perioperative complication, attributing up to 9% of all anesthesia-related deaths. However, there is currently no practical, noninvasive bedside test to determine gastric content and volume in the perioperative period. Methods:The current study evaluates the feasibility of using bedside ultrasonography for assessing gastric content and volume. In the pilot phase, 18 healthy volunteers were examined to assess the gastric antrum, body, and fundus in cross-section in five prandial states: fasting and after ingestion of 250 mL of water, 500 mL of water, 500 mL of effervescent water, and a solid meal. In the phase II study, the authors concentrated on ultrasound examination of the gastric antrum in 36 volunteers for whom regression analysis was used to determine the correlation between gastric volume and antral cross-sectional area. Results:The gastric antrum provided the most reliable quantitative information for gastric volume. The antral cross-sectional area correlated with volumes of up to 300 mL in a close-to-linear fashion, particularly when subjects were in the right lateral decubitus position. Sonographic assessment of the gastric antrum and body provides qualitative information about gastric content (empty or not empty) and its nature (gas, fluid, or solid). The fundus was the gastric area least amenable to image and measure. Conclusions:Our preliminary results suggest that bedside two-dimensional ultrasonography can be a useful noninvasive tool to determine gastric content and volume.

Preoperative use of statins is associated with reduced early delirium rates after cardiac surgery.

Background:Delirium is an acute deterioration of brain function characterized by fluctuating consciousness and an inability to maintain attention. Use of statins has been shown to decrease morbidity and mortality after major surgical procedures. The objective of this study was to determine an association between preoperative administration of statins and postoperative delirium in a large prospective cohort of patients undergoing cardiac surgery with cardiopulmonary bypass. Methods:After Institutional Review Board approval, data were prospectively collected on consecutive patients undergoing cardiac surgery with cardiopulmonary bypass from April 2005 to June 2006 in an academic hospital. All patients were screened for delirium during their hospitalization using the Confusion Assessment Method in the intensive care unit. Multivariable logistic regression analysis was used to identify independent perioperative predictors of delirium after cardiac surgery. Statins were tested for a potential protective effect. Results:Of the 1,059 patients analyzed, 122 patients (11.5%) had delirium at any time during their cardiovascular intensive care unit stay. Administration of statins had a protective effect, reducing the odds of delirium by 46%. Independent predictors of postoperative delirium included older age, preoperative depression, preoperative renal dysfunction, complex cardiac surgery, perioperative intraaortic balloon pump support, and massive blood transfusion. The model was reliable (Hosmer-Lemeshow test, P = 0.3) and discriminative (area under receiver operating characteristic curve = 0.77). Conclusions:Preoperative administration of statins is associated with the reduced risk of postoperative delirium after cardiac surgery with cardiopulmonary bypass.

Gastric sonography in the fasted surgical patient: a prospective descriptive study.

BACKGROUND: Aspiration pneumonia remains a serious anesthetic-related complication. A reliable diagnostic tool to assess gastric volume is currently lacking. We recently demonstrated that gastric sonography can provide reliable qualitative and quantitative information about gastric content and volume in healthy volunteers. In the current study, we performed a prospective qualitative and quantitative analysis of the gastric antrum in 200 fasted patients undergoing elective surgery. METHODS: A standardized gastric scanning protocol was applied before anesthetic induction. Patients were classified following a 3-point grading system based solely on qualitative sonographic assessment of the antrum in the supine and right lateral decubitus positions. RESULTS: Eighty-six patients were classified as grade 0 (empty antrum); 107 patients as grade 1 (minimal fluid volume detected only in the right lateral decubitus position); and 7 patients were classified as grade 2 (antrum clearly distended with fluid visible in both supine and lateral positions). The 3-point grading system correlated with total gastric fluid volume as predicted by a previously reported mathematical model. Essentially grade 0 corresponds to a completely empty stomach, grade 1 corresponds to negligible fluid volumes (16 ± 36 mL) within normal ranges expected for fasted patients, and grade 2 correlates with significantly higher predicted gastric fluid volumes (180 ± 83 mL) beyond previously reported “safe” limits. One patient with a grade 2 antrum had an episode of significant regurgitation of gastric contents on emergence from anesthesia. CONCLUSION: We propose a 3-point grading system based exclusively on qualitative sonographic assessment of the gastric antrum that correlates well with predicted gastric volume. This grading system could be a promising “biomarker” to assess perioperative aspiration risk. Before it can be applied widely to clinical practice, this diagnostic tool needs to be further validated and characterized.

Adding Gabapentin to a multimodal regimen does not reduce acute pain, opioid consumption or chronic pain after total hip arthroplasty

Background: Gabapentin (GPN) is effective in reducing post‐operative pain and opioid consumption, but its effects with regional anesthesia for total hip arthroplasty (THA) are not known. We designed this study to determine whether (1) gabapentin administration reduces pain and opioid use after THA using a multimodal analgesic regimen including spinal anesthesia; (2) pre‐operative administration of gabapentin is more effective than post‐operative administration.

Acute Surgical Anemia Influences the Cardioprotective Effects of β-Blockade: A Single-center, Propensity-matched Cohort Study

Background:Despite decreasing cardiac events, perioperative &bgr;-blockade also increases perioperative stroke and mortality. Major bleeding and/or hypotension are independently associated with these outcomes. To investigate the hypothesis that &bgr;-blockade limits the cardiac reserve to compensate for acute surgical anemia, the authors examined the relationship between cardiac events and acute surgical anemia in patients with and without &bgr;-blockade. Methods:The records of all noncardiac, nontransplant surgical patients between March 2005 and June 2006 were retrospectively retrieved. The primary outcome was a composite that comprised myocardial infarction, nonfatal cardiac arrest, and in-hospital mortality (major adverse cardiac event). The lowest recorded hemoglobin in the first 3 days defined nadir hemoglobin. Propensity scores estimating the probability of receiving a perioperative &bgr;-blocker were used to match (1:1) patients who did or did not receive &bgr;-blockers postoperatively. The relationship between nadir hemoglobin and major adverse cardiac event was then assessed. Results:This analysis identified 4,387 patients in whom nadir hemoglobin could be calculated; 1,153 (26%) patients were administered &bgr;-blockers within the first 24 h of surgery. Propensity scores created 827 matched pairs that were well balanced for all measured confounders. Major adverse cardiac event occurred in 54 (6.5%) &bgr;-blocked patients and in 25 (3.0%) &bgr;-blocker naive patients (relative risk 2.38; 95% CI 1.43–3.96; P = 0.0009). The restricted cubic spline relationship demonstrated that this difference was restricted to those patients in whom the hemoglobin decrease exceeded 35% of the baseline value. Conclusions:&bgr;-Blocked patients do not seem to tolerate surgical anemia when compared with patients who are naive to &bgr;-blockers. Prospective studies are required to validate these findings.

Validation of a mathematical model for ultrasound assessment of gastric volume by gastroscopic examination.

INTRODUCTION:Pulmonary aspiration of gastric contents is a serious perioperative complication. Previous models of ultrasound gastric volume assessment are preliminary and have not been validated by an external “gold standard.” In the present study we propose a more accurate model based on prospective data obtained from 108 patients undergoing bedside gastric sonography and upper gastrointestinal endoscopy (UGE). METHODS:Patients undergoing elective UGE were randomized to ingest one of 6 predetermined volumes of apple juice after an 8-hour fasting period. A cross-sectional area of the antrum in the right lateral decubitus position (Right lat CSA) was measured by a blinded sonographer following a standardized scanning protocol. Gastric fluid was subsequently suctioned under gastroscopic vision during UGE performed by a blinded gastroenterologist and measured to the nearest milliliter. RESULTS:Data from 108 patients suggest that a previously reported model tends to overestimate gastric volume particularly at low volume states. A new best fit mathematical model to predict gastric fluid volume based on measurements of Right lat CSA is presented. This new model built on a more accurate gold standard can be used to estimate gastric volumes from 0 to 500 mL, in nonpregnant adults with body mass index < 40 kg/m2. CONCLUSIONS:We report a new prediction model to assess gastric fluid volume using standard 2-dimentional bedside ultrasound that has several advantages over previously reported models.

Metoprolol Reduces Cerebral Tissue Oxygen Tension after Acute Hemodilution in Rats

Background:Perioperative &bgr;-blockade and anemia are independent predictors of increased stroke and mortality by undefined mechanisms. This study investigated the effect of &bgr;-blockade on cerebral tissue oxygen delivery in an experimental model of blood loss and fluid resuscitation (hemodilution). Methods:Anesthetized rats were treated with metoprolol (3 mg · kg−1) or saline before undergoing hemodilution with pentastarch (1:1 blood volume exchange, 30 ml · kg−1). Outcomes included cardiac output, cerebral blood flow, and brain (PBrO2) and kidney (PKO2) tissue oxygen tension. Hypoxia inducible factor-1&agr; (HIF-1&agr;) protein levels were assessed by Western blot. Systemic catecholamines, erythropoietin, and angiotensin II levels were measured. Results:Hemodilution increased heart rate, stroke volume, cardiac output (60%), and cerebral blood flow (50%), thereby maintaining PBrO2 despite an approximately 50% reduction in blood oxygen content (P < 0.05 for all). By contrast, PKO2 decreased (50%) under the same conditions (P < 0.05). &bgr;-blockade reduced baseline heart rate (20%) and abolished the compensatory increase in cardiac output after hemodilution (P < 0.05). This attenuated the cerebral blood flow response and reduced PBrO2 (50%), without further decreasing PKO2. Cerebral HIF-1&agr; protein levels were increased in &bgr;-blocked hemodiluted rats relative to hemodiluted controls (P < 0.05). Systemic catecholamine and erythropoietin levels increased comparably after hemodilution in both groups, whereas angiotensin II levels increased only after &bgr;-blockade and hemodilution. Conclusions:Cerebral tissue oxygen tension is preferentially maintained during hemodilution, relative to the kidney, despite elevated systemic catecholamines. Acute &bgr;-blockade impaired the compensatory cardiac output response to hemodilution, resulting in a reduction in cerebral tissue oxygen tension and increased expression of HIF-1&agr;.

Spatial and temporal mapping of heterogeneity in liposome uptake and microvascular distribution in an orthotopic tumor xenograft model

Existing paradigms in nano-based drug delivery are currently being challenged. Assessment of bulk tumor accumulation has been routinely considered an indicative measure of nanomedicine potency. However, it is now recognized that the intratumoral distribution of nanomedicines also impacts their therapeutic effect. At this time, our understanding of the relationship between the bulk (i.e., macro-) tumor accumulation of nanocarriers and their intratumoral (i.e., micro-) distribution remains limited. Liposome-based drug formulations, in particular, suffer from diminished efficacy in vivo as a result of transport-limiting properties, combined with the heterogeneous nature of the tumor microenvironment. In this report, we perform a quantitative image-based assessment of macro- and microdistribution of liposomes. Multi-scalar assessment of liposome distribution was enabled by a stable formulation which co-encapsulates an iodinated contrast agent and a near-infrared fluorescence probe, for computed tomography (CT) and optical microscopy, respectively. Spatio-temporal quantification of tumor uptake in orthotopic xenografts was performed using CT at the bulk tissue level, and within defined sub-volumes of the tumor (i.e., rim, periphery and core). Tumor penetration and relative distribution of liposomes were assessed by fluorescence microscopy of whole tumor sections. Microdistribution analysis of whole tumor images exposed a heterogeneous distribution of both liposomes and tumor vasculature. Highest levels of liposome uptake were achieved and maintained in the well-vascularized tumor rim over the study period, corresponding to a positive correlation between liposome and microvascular density. Tumor penetration of liposomes was found to be time-dependent in all regions of the tumor however independent of location in the tumor. Importantly, a multi-scalar comparison of liposome distribution reveals that macro-accumulation in tissues (e.g., blood, whole tumor) may not reflect micro-accumulation levels present within specific regions of the tumor as a function of time.