A mitochondrial protein compendium elucidates complex I disease biology.

Mitochondria are complex organelles whose dysfunction underlies a broad spectrum of human diseases. Identifying all of the proteins resident in this organelle and understanding how they integrate into pathways represent major challenges in cell biology. Toward this goal, we performed mass spectrometry, GFP tagging, and machine learning to create a mitochondrial compendium of 1098 genes and their protein expression across 14 mouse tissues. We link poorly characterized proteins in this inventory to known mitochondrial pathways by virtue of shared evolutionary history. Using this approach, we predict 19 proteins to be important for the function of complex I (CI) of the electron transport chain. We validate a subset of these predictions using RNAi, including C8orf38, which we further show harbors an inherited mutation in a lethal, infantile CI deficiency. Our results have important implications for understanding CI function and pathogenesis and, more generally, illustrate how our compendium can serve as a foundation for systematic investigations of mitochondria.

A parallel microfluidic flow cytometer for high-content screening.

A parallel microfluidic cytometer (PMC) uses a high-speed scanning photomultiplier-based detector to combine low-pixel-count, one-dimensional imaging with flow cytometry. The 384 parallel flow channels of the PMC decouple count rate from signal-to-noise ratio. Using six-pixel one-dimensional images, we investigated protein localization in a yeast model for human protein misfolding diseases and demonstrated the feasibility of a nuclear-translocation assay in Chinese hamster ovary (CHO) cells expressing an NFκB-EGFP reporter.

Deep ultraviolet mapping of intracellular protein and nucleic acid in femtograms per pixel.

By using imaging spectrophotometry with paired images in the 200- to 280-nm wavelength range, we have directly mapped intracellular nucleic acid and protein distributions across a population of Chinese hamster ovary (CHO-K1) cells. A broadband 100× objective with a numerical aperture of 1.2 NA (glycerin immersion) and a novel laser-induced-plasma point source generated high-contrast images with short (∼100 ms) exposures and a lateral resolution nearing 200 nm that easily resolves internal organelles. In a population of 420 CHO-K1 cells and 477 nuclei, we found a G1 whole-cell nucleic acid peak at 26.6 pg, a nuclear-isolated total nucleic acid peak at 11.4 pg, and, as inferred by RNase treatment, a G1 total DNA mass of 7.4 pg. At the G1 peak, we found a whole-cell protein mass of 95.6 pg, and a nuclear-isolated protein mass of 39.3 pg. An algorithm for protein quantification that senses peptide-bond (220-nm) absorbance was found to have a higher signal-to-noise ratio and to provide more reliable nucleic acid and protein determinations when compared to more classical 280/260-nm algorithms when used for intracellular mass mapping. Using simultaneous imaging with common nuclear stains (Hoechst 33342, Syto-14, and Sytox Orange), we have compared staining patterns to deep-UV images of condensed chromatin and have confirmed bias of these common nuclear stains related to nuclear packaging. The approach allows absolute mass measurements with no special sample preparation or staining. It can be used in conjunction with normal fluorescence microscopy and with relatively modest modification of the microscope.

Insulin causes fatty acid transport protein translocation and enhanced fatty acid uptake in adipocytes.

Fatty acid uptake into 3T3 L1 adipocytes is predominantly transporter mediated. Here we show that, during 3T3 L1 adipocyte differentiation, expression of fatty acid transport proteins (FATPs) 1 and 4 is induced. Using subcellular membrane fractionation and immunofluorescence microscopy, we demonstrate that, in adipocytes, insulin induces plasma membrane translocation of FATPs from an intracellular perinuclear compartment to the plasma membrane. This translocation was observed within minutes of insulin treatment and was paralleled by an increase in long chain fatty acid (LCFA) uptake. In contrast, treatment with TNF-alpha inhibited basal and insulin-induced LCFA uptake and reduced FATP1 and -4 levels. Thus, hormonal regulation of FATP activity may play an important role in energy homeostasis and metabolic disorders such as type 2 diabetes.

Macrophage podosomes assemble at the leading lamella by growth and fragmentation.

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with beta-actin-ECFP and L-fimbrin-EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.

Visualization of replication initiation and elongation in Drosophila.

Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2-7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. We utilized this system to demonstrate that the pre-replication complex component, double-parked protein/cell division cycle 10-dependent transcript 1, is not only necessary for proper MCM2-7 localization, but, unexpectedly, is present during elongation.

Prognosis after late relapse following epilepsy surgery.

OBJECTIVES: To assess prognosis after late relapse in patients who are seizure free for the first 5 years after epilepsy surgery. METHODS: Patients who were seizure free for the first 5 years after resective epilepsy surgery were included. Date of first seizure recurrence, current seizure status, medication, age, and type of surgery were prospectively registered. Non-parametric statistics were used. RESULTS: One hundred and fifty-nine patients were studied. Thirty-two had at least one recurrent seizure. Time to event analysis showed an annual relapse rate of 4% between years 5 and 10 after surgery. At study termination, 143 of 159 patients (89.9%) were in terminal remission. For 30 patients with late relapse and at least 1-year follow-up thereafter, 53% were in terminal remission and 30% had experienced only rare or nocturnal seizures. Medication use was not associated either with likelihood of relapse or entering remission after relapse. CONCLUSIONS: Patients who are seizure free for the first 5 years after epilepsy surgery remain at risk for seizure recurrence. These relapses are often isolated events, and the long-term prognosis after relapse is often good. Relapse rates were similar in patients on and off AEDs, but the relation between AED taper and relapse is uncertain since patient groups may not be similar.

Antiepileptic drugs and relapse after epilepsy surgery.

PURPOSE: To evaluate whether the postoperative, antiepileptic drug (AED) regimen influences seizure recurrence after anterior temporal lobectomy when considering the putative mechanism of action and possible neuroprotective effects. METHODS: This was a retrospective study. Patients who had an anterior temporal lobectomy for refractory epilepsy, whose preoperative MRI indicated mesial temporal sclerosis, were included. Postoperative AED regimens were compared with regard to seizure-outcome, considering the putative mechanism of action (sodium channel blockers, non-sodium channel blockers, and mixed mechanisms) or possible neuroprotective effect (levetiracetam, topiramate, tiagabine and zonisamide versus others). Time-to-event (first seizure after surgery) analysis was used to produce a Kaplan-Meier estimate of seizure recurrence, and groups were compared using Cox proportional hazard analysis. RESULTS: 226 patients (103 males and 123 females; mean age 42 +/- 11 years) were studied. The rates of postoperative seizure recurrence were not significantly different between the three groups regardless of the use of AEDs with different mechanisms of action (p = 0.23). Fifty patients were receiving possibly neuroprotective AEDs and 176 patients were not. Rates of seizure recurrence were not significantly different between these two groups either (p = 0.11). The differences between one-year seizure-free rates were not significant when we compared levetiracetam versus phenytoin or carbamazepine. DISCUSSION: There appeared to be no advantage or disadvantage to either prescribing drugs with different mechanisms of action or using drugs with possible neuroprotective effect after temporal lobectomy. Prospective studies with larger sample sizes may be of benefit to further explore this issue.

Linking microscopy and high content screening in large-scale biomedical research.

Microscopy has been a cornerstone of discovery in the academic life sciences for more than 100 yr. This comes from a unique ability to provide extremely rich information of biological structure and dynamics. The advent of digital imaging and machine vision has brought within itself the ability to collect images more easily and critically, the ability to measure objects and intensities within images. Although many continue to use microscopy in a qualitative manner, the analytical capabilities afforded by machine vision are increasingly being applied to basic cell biology and biomedical research. Scalable quantitative imaging technology might enable scientists and engineers to determine structure, dynamics, and function of entire biological systems rather than individual molecules or pathways. This chapter will provide an overview of early efforts in the academic community to apply high content screening to the study of biological systems.

The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit.

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.

Structure and dynamics of macrophage podosomes.

Podosomes are short-lived, dynamic actin-rich adhesions classically formed in macrophages, osteoclasts and Src-transformed fibroblasts. Though related to the more commonly studied focal adhesions, sharing several structural and regulatory proteins, podosomes have distinct functional, structural, and dynamic characteristics. Here, we discuss current understanding of the function of podosomes in disparate cell types and how this relates to their structure and dynamics.

Parallel imaging microfluidic cytometer.

By adding an additional degree of freedom from multichannel flow, the parallel microfluidic cytometer (PMC) combines some of the best features of fluorescence-activated flow cytometry (FCM) and microscope-based high-content screening (HCS). The PMC (i) lends itself to fast processing of large numbers of samples, (ii) adds a 1D imaging capability for intracellular localization assays (HCS), (iii) has a high rare-cell sensitivity, and (iv) has an unusual capability for time-synchronized sampling. An inability to practically handle large sample numbers has restricted applications of conventional flow cytometers and microscopes in combinatorial cell assays, network biology, and drug discovery. The PMC promises to relieve a bottleneck in these previously constrained applications. The PMC may also be a powerful tool for finding rare primary cells in the clinic. The multichannel architecture of current PMC prototypes allows 384 unique samples for a cell-based screen to be read out in ∼6-10 min, about 30 times the speed of most current FCM systems. In 1D intracellular imaging, the PMC can obtain protein localization using HCS marker strategies at many times for the sample throughput of charge-coupled device (CCD)-based microscopes or CCD-based single-channel flow cytometers. The PMC also permits the signal integration time to be varied over a larger range than is practical in conventional flow cytometers. The signal-to-noise advantages are useful, for example, in counting rare positive cells in the most difficult early stages of genome-wide screening. We review the status of parallel microfluidic cytometry and discuss some of the directions the new technology may take.

A cell profiling framework for modeling drug responses from HCS imaging.

The authors present an unsupervised, scalable, and interpretable cell profiling framework that is compatible with data gathered from high-content screening. They demonstrate the effectiveness of their framework by modeling drug differential effects of IC-21 macrophages treated with microtubule and actin disrupting drugs. They identify significant features of cell phenotypes for unsupervised learning based on maximum relevancy and minimum redundancy criteria. A 2-stage clustering approach annotates, clusters cells, and then merges them together to form super-clusters. An interpretable cell profile consisting of super-cluster proportions profiled at each drug treatment, concentration, or duration is obtained. Differential changes in super-cluster profiles are the basis for understanding the drug's differential effect and biology. The authors' method is validated by significant chi-squared statistics obtained from similar drug-treated super-cluster profiles from a 5-fold cross-validation. In addition, drug profiles of 2 microtubule drugs with equivalent mechanisms of action are statistically similar. Several distinct trends are identified for the 5 cytoskeletal drugs profiled under different conditions.

Recurrence in nonseminomatous germ cell testis tumor patients with no viable tumor at postchemotherapy retroperitoneal lymph node dissection.

OBJECTIVES: To determine disease-related outcomes in metastatic testis cancer patients with absence of viable cancer in the postchemotherapy retroperitoneal lymph node dissection (PC-RPLND) specimen and determine whether clinical variables can help predict disease progression. METHODS: Between 1980 and 2003, 195 patients had no viable tumor at the time of PC-RPLND. We retrospectively reviewed their medical records for pertinent clinical and treatment-related outcomes. At a median follow-up of 45 months (range, 6 to 236 months), 35 patients (18%) developed recurrences, and 18 (9%) died of disease. RESULTS: On multivariate analysis, predictors of recurrence-free survival in patients with no viable tumor were advanced clinical stage (P = 0.01) and poor-risk International Germ Cell Consensus Classification (IGCCC) group (P = 0.01), whereas predictors of disease-specific survival included an elevated serum beta-human chorionic gonadotropin (hCG) level before PC-RPLND (P = 0.002), pathologic diameter of the retroperitoneal mass (P = 0.05), and postoperative recurrence (P <0.0001). An hCG level greater than 1.2 mIU/mL before PC-RPLND trended toward statistical significance (P = 0.07), and pathologic diameter of the retroperitoneal mass greater than 2.5 cm was statistically significant (P = 0.05) in predicting a poorer disease-specific survival. CONCLUSIONS: Patients with no viable tumor at PC-RPLND remain at risk of recurrence. Several clinical variables, including advanced clinical stage, poor-risk IGCCC group, preoperative serum hCG level, diameter of the retroperitoneal mass on pathology, and postoperative recurrence, help better define which patients are at risk.

Viable germ cell tumor at postchemotherapy retroperitoneal lymph node dissection: can we predict patients at risk of disease progression?

BACKGROUND: Patients with viable tumor at time of postchemotherapy retroperitoneal lymph node dissection (PC-RPLND) are at an increased risk of disease progression. The objective of the current study was to determine the clinical variables that predict this adverse outcome. METHODS: Between 1980 and 2003, 236 patients with testicular cancer underwent PC-RPLND, 41 of whom (17%) were found to have viable tumor. The authors retrospectively reviewed the patients' medical records for pertinent clinical and treatment-related outcomes. At a median follow-up of 3.9 years, 18 patients (44%) had developed disease recurrence and 12 patients (29%) had died of disease. RESULTS: The group of patients who developed postoperative disease recurrence had a larger median dimension of the retroperitoneal mass (7.0 cm and 3.5 cm, respectively; P = .03). The use of adjuvant chemotherapy after PC-RPLND was less common in those patients developing postoperative disease recurrence (P = .06). On multivariate analysis, patients classified as being at intermediate or poor risk according to the International Germ Cell Consensus Classification (IGCCC) had a poorer recurrence-free survival (P = .006 and P = .07, respectively). On multivariate analysis, predictors of disease-specific survival (DSS) included an elevated alpha-fetoprotein (AFP) level before PC-RPLND (P = .003) and postoperative disease recurrence (P = .02). A serum AFP level >5.3 ng/mL before PC-RPLND was found to be predictive of a poorer DSS (P = .0007). CONCLUSIONS: Patients with viable tumor at the time of PC-RPLND are at an increased risk of disease progression. Clinical variables including classification as intermediate or poor IGCCC risk, a preoperative serum AFP level >5.3 ng/mL, and postoperative disease recurrence help to better define those patients who are at risk of future adverse outcomes.

Recurrence pattern and proposed surveillance protocol following post-chemotherapy retroperitoneal lymph node dissection.

PURPOSE: We evaluated the recurrence pattern in patients with nonseminomatous germ cell tumors treated with post-chemotherapy retroperitoneal lymph node dissection and determined the optimal surveillance strategy in these patients. MATERIALS AND METHODS: Between 1980 and 2003, 236 patients with clinical stage IIA-III nonseminomatous germ cell tumors underwent post-chemotherapy retroperitoneal lymph node dissection. Patients with increased preoperative tumor markers (alpha-fetoprotein greater than 15 ng/ml and/or beta-human chorionic gonadotropin greater than 2.2 U/ml) were excluded from study resulting in 198 patients for analysis. We retrospectively reviewed medical records for pertinent clinical and treatment related outcomes. In our patient population recurrence developed in 45 (23%) patients and 22 (11%) died of disease at a median followup of 41 months (range 6 to 250) after retroperitoneal lymph node dissection. RESULTS: The clinical stage of testis cancer was IIA in 17, IIB in 49, IIC in 83 and III in 49 patients. Of the 45 patients with postoperative recurrence, 16 had concomitant multiple sites of recurrence with a total of 64 sites reported. Of the cases of recurrence 21 (46.7%) were in those of clinical stage III, 18 (40%) stage IIC and 6 (11.8%) stage IIB disease. The most frequent site of recurrence was the chest (32, 49%), followed by the abdomen (14, 22%), supraclavicular lymph nodes (8, 13%), brain (5, 8%) and other sites (5, 8%). CONCLUSIONS: Based on the recurrence pattern we propose stage specific surveillance guidelines for the followup of patients after post-chemotherapy retroperitoneal lymph node dissection. These guidelines help identify patients at high risk for disease progression and, thus, requiring more stringent postoperative followup.

Chylous ascites after post-chemotherapy retroperitoneal lymph node dissection: review of the M. D. Anderson experience.

PURPOSE: We determined the clinical presentation, risk factors and optimal treatment of chylous ascites that develop after retroperitoneal lymph node dissection in patients with testicular cancer. MATERIALS AND METHODS: We retrospectively reviewed the records of 329 patients who underwent post-chemotherapy retroperitoneal lymph node dissection at our institution, of whom 23 (7%) had chylous ascites postoperatively. Clinical and pathological parameters were entered into a database. RESULTS: Mean patient age at chylous ascites presentation was 32.1 years. On univariate and multivariate logistic regression analyses increasing amounts of preoperative chemotherapy (OR 1.24) and intraoperative blood loss (OR 1.33) were predictive of chylous ascites. The clinical presentation of chylous ascites consisted of abdominal fullness and distention in all patients. Initial treatment was paracentesis alone or combined with total parenteral nutrition in 77% of patients. An abdominal drain was used for persistent ascites in 10 patients. In patients treated conservatively the rate of resolution of chylous ascites was 77%. Only 23% of patients required peritoneovenous shunt placement. However, shunt use was associated with an 80% surgical revision rate. CONCLUSIONS: Conservative treatment resolves most cases of postoperative chylous ascites. An abdominal catheter drain should be considered for significant or recurring chylous ascites. When a peritoneovenous shunt is required, it may be needed for an extensive period for resolution and there are significant complications associated with its use. Increasing amounts of preoperative chemotherapy and operative blood loss raise the likelihood of chylous ascites.

Viable malignant germ cell tumor in the postchemotherapy retroperitoneal lymph node dissection specimen: can it be predicted using clinical parameters?

BACKGROUND: The presence of viable tumor in the surgical specimen after postchemotherapy retroperitoneal lymph node dissection (PC-RPLND) is associated with an increased risk of disease progression. The objective of this study was to determine whether the presence of viable tumor in the surgical specimen could be predicted. METHODS: Between 1980 and 2003, 236 patients underwent PC-RPLND for clinical Stage IIA or III nonseminomatous germ cell tumors (NSGCT). The authors retrospectively reviewed the medical records of those patients for pertinent clinical and treatment-related outcomes. A multivariate logistic regression analysis was used to evaluate whether clinical parameters were capable of predicting the presence of viable tumor in the surgical specimen. RESULTS: International Germ Cell Consensus Classification (IGCCC) risk categories could be assigned to 218 patients, with 101 patients in the good-risk category, 32 patients in the intermediate-risk category, and 85 patients in the poor-risk category. The incidence of viable tumor in the good-risk, intermediate-risk, and poor-risk categories was similar (17.8%, 15.6%, and 15.3%, respectively); however, the risk categories predicted disease-specific and recurrence-free survival (P = .022 and P < .0001, respectively). On multivariate analysis, an elevated serum alpha-fetoprotein (AFP) level prior to PC-RPLND (P = .05) and the size of the retroperitoneal mass on pathology review (P = .02) were predictive of viable tumor in the surgical specimen. CONCLUSIONS: Although IGCCC risk categories were correlated with disease-related outcomes, the risk groups had similar incidences of viable tumor. Elevated serum AFP levels prior to surgery and the size of the retroperitoneal mass in the resected specimen may help to predict viable tumor in the PC-RPLND specimen.

Predictors of outcome in patients undergoing postchemotherapy retroperitoneal lymph node dissection for testicular cancer.

BACKGROUND: The management of metastatic nonseminomatous germ cell tumors (NSGCT) frequently consists of systemic chemotherapy followed by retroperitoneal lymph node dissection (PC-RPLND). The aim of the present study was to evaluate the authors' PC-RPLND experience and identify predictors of outcome in these patients. METHODS: Between 1980 and 2003, 236 patients with clinical Stage IIA-III NSGCT underwent PC-RPLND. Their medical records were retrospectively reviewed for pertinent clinical and treatment-related outcomes. The 5-year disease-specific and recurrence-free survival was 85% and 75%, respectively, with the median length of follow-up after RPLND 45 months (6-250 months). RESULTS: The median age of patients at diagnosis was 28 years, with all patients receiving systemic chemotherapy (median of 5 cycles) before RPLND. On multivariate analysis, predictors of poorer disease-specific survival (DSS) included systemic symptoms at presentation (P = .05), elevated pre-RPLND serum alpha fetoprotein (AFP, P = .006) and beta-human chorionic gonadotropin (HCG, P = .004), postoperative complications (P = .03), and recurrence (P < .0001). Predictors of poorer recurrence-free survival (RFS) included advanced clinical stage (IIC-III, P = .001) and presence of viable tumor in the RPLND specimen (P = .03). A pre-RPLND serum AFP > 9 ng/mL and HCG > 4.1 mIU/mL were found to predict a worse DSS (P = .03 and .03, respectively). CONCLUSIONS: In patients undergoing PC-RPLND, preoperative tumor markers and the occurrence of postoperative complications or recurrence are predictive of poorer DSS. Advanced clinical stage and viable tumor in the surgical specimen predict worse RFS.