CURE on yeast genes of unknown function increases students' bioinformatics proficiency and research confidence.

Course-based undergraduate research experiences (CUREs) can reduce barriers to research opportunities while increasing student knowledge and confidence. However, the number of widely adopted, easily transferable CUREs is relatively small. Here, we describe a CURE aimed at determining the function of poorly characterized Saccharomyces cerevisiae genes. More than 20 years after sequencing of the yeast genome, nearly 10% of open reading frames (ORFs) still have at least one uncharacterized Gene Ontology (GO) term. We refer to these genes as "ORFans" and formed a consortium aimed at assigning functions to them. Specifically, over 70 faculty members attended summer workshops to learn the bioinformatics workflow and basic laboratory techniques described herein. Ultimately, this CURE was adapted for implementation at 34 institutions, resulting in over 1,300 students conducting course-based research on ORFans. Pre-/post-tests confirmed that students gained both (i) an understanding of gene ontology and (ii) knowledge regarding the use of bioinformatics to assign gene function. After using these data to craft their own hypotheses, then testing their predictions by constructing and phenotyping deletion strains, students self-reported significant gains in several areas, including computer modeling and exposure to a project where no one knows the outcome. Interestingly, most net gains self-reported by ORFan Gene Project participants were greater than published findings for CUREs assessed with the same survey instrument. The surprisingly strong impact of this CURE may be due to the incoming lack of experience of ORFan Project participants and/or the independent thought required to develop testable hypotheses from complex data sets.

DMSO reduces the cytotoxicity of anticancer ruthenium complex KP1019 in yeast.

Low solubility in aqueous solutions is a significant limitation of the otherwise promising anticancer ruthenium complex KP1019. In laboratory studies, this challenge is often overcome by using DMSO to help drive the drug into solution. Since DMSO was previously shown to alter the bioactivity of platinum-based chemotherapeutics, here we examine DMSO's effects on KP1019. Using Saccharomyces cerevisiae as a model organism, we apply multiple measures of growth inhibition to demonstrate that DMSO reduces the drug's toxicity. This reduction in bioactivity correlates with spectrophotometric changes consistent with DMSO-dependent increases in the stability of the KP1019 pro-drug. The impact of DMSO on the biology and chemistry of KP1019 suggests this solvent should not be used in studies of this and similar anticancer ruthenium complexes.

Proteomic analysis of the S. cerevisiae response to the anticancer ruthenium complex KP1019.

Like platinum-based chemotherapeutics, the anticancer ruthenium complex indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(iii)], or KP1019, damages DNA, induces apoptosis, and causes tumor regression in animal models. Unlike platinum-based drugs, KP1019 showed no dose-limiting toxicity in a phase I clinical trial. Despite these advances, the mechanism(s) and target(s) of KP1019 remain unclear. For example, the drug may damage DNA directly or by causing oxidative stress. Likewise, KP1019 binds cytosolic proteins, suggesting DNA is not the sole target. Here we use the budding yeast Saccharomyces cerevisiae as a model in a proteomic study of the cellular response to KP1019. Mapping protein level changes onto metabolic pathways revealed patterns consistent with elevated synthesis and/or cycling of the antioxidant glutathione, suggesting KP1019 induces oxidative stress. This result was supported by increased fluorescence of the redox-sensitive dye DCFH-DA and increased KP1019 sensitivity of yeast lacking Yap1, a master regulator of the oxidative stress response. In addition to oxidative and DNA stress, bioinformatic analysis revealed drug-dependent increases in proteins involved ribosome biogenesis, translation, and protein (re)folding. Consistent with proteotoxic effects, KP1019 increased expression of a heat-shock element (HSE) lacZ reporter. KP1019 pre-treatment also sensitized yeast to oxaliplatin, paralleling prior research showing that cancer cell lines with elevated levels of translation machinery are hypersensitive to oxaliplatin. Combined, these data suggest that one of KP1019's many targets may be protein metabolism, which opens up intriguing possibilities for combination therapy.

DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae.

Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage--including that induced by many anticancer drugs--results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events.

Le Fort III distraction using rotation advancement of the midface in patients with cleft lip and palate.

BACKGROUND: Patients with cleft lip and palate demonstrate a spectrum of maxillary growth deficiencies. The purpose of this study was to review the authors' experience in the treatment of midface hypoplasia in nonsyndromic cleft lip-cleft palate patients using rotation advancement of the midface with Le Fort III distraction. METHODS: A retrospective chart review was conducted to include all patients with nonsyndromic cleft lip and/or cleft palate who underwent Le Fort III rotation advancement of the midface from 1999 to 2011. Along with standard outcome measures, Amira imaging software was used to perform surface analysis on the last five consecutive patients in this series. RESULTS: Forty-three consecutive patients met inclusion criteria. Diagnoses included unilateral complete cleft lip and palate (n = 25) and bilateral complete cleft lip and palate (n = 18). Average distraction distance measured 10.1 mm at the level of the zygoma (range, 6 to 15 mm). Preoperative and 6-month postoperative sella, nasion, A point angles measured 76.3 and 81.8 degrees; whereas sella, nasion, B point angles measured 79.9 and 78.7 degrees, respectively. Preoperative and 6-month postoperative overjet measured -5.4 and 3.2 mm, whereas overbite measured 1.9 and 1.1 mm, respectively. Six patients (13 percent) developed pseudorelapse, with five patients going on to have subsequent Le Fort I advancement at an average of 7 years after distraction (range, 2 to 11 years). CONCLUSIONS: Le Fort III rotation advancement of the midface addresses severe midface deficiencies in select patients. Whereas older techniques target occlusal correction alone, extending osteotomies to the Le Fort III level allows occlusal correction along with improvements in malar and nasal projection required to achieve facial harmony in this group of patients. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

The anticancer ruthenium complex KP1019 induces DNA damage, leading to cell cycle delay and cell death in Saccharomyces cerevisiae.

The anticancer ruthenium complex trans-[tetrachlorobis(1H-indazole)ruthenate(III)], otherwise known as KP1019, has previously been shown to inhibit proliferation of ovarian tumor cells, induce DNA damage and apoptosis in colon carcinoma cells, and reduce tumor size in animal models. Notably, no dose-limiting toxicity was observed in a Phase I clinical trial. Despite these successes, KP1019's precise mechanism of action remains poorly understood. To determine whether Saccharomyces cerevisiae might serve as an effective model for characterizing the cellular response to KP1019, we first confirmed that this drug is internalized by yeast and induces mutations, cell cycle delay, and cell death. We next examined KP1019 sensitivity of strains defective in DNA repair, ultimately showing that rad1Δ, rev3Δ, and rad52Δ yeast are hypersensitive to KP1019, suggesting that nucleotide excision repair (NER), translesion synthesis (TLS), and recombination each play a role in drug tolerance. These data are consistent with published work showing that KP1019 causes interstrand cross-links and bulky DNA adducts in mammalian cell lines. Published research also showed that mammalian cell lines resistant to other chemotherapeutic agents exhibit only modest resistance, and sometimes hypersensitivity, to KP1019. Here we report similar findings for S. cerevisiae. Whereas gain-of-function mutations in the transcription activator-encoding gene PDR1 are known to increase expression of drug pumps, causing resistance to structurally diverse toxins, we now demonstrate that KP1019 retains its potency against yeast carrying the hypermorphic alleles PDR1-11 or PDR1-3. Combined, these data suggest that S. cerevisiae could serve as an effective model system for identifying evolutionarily conserved modulators of KP1019 sensitivity.

Complications of mandibular distraction osteogenesis.

BACKGROUND: Mandibular hypoplasia, retrognathia, and micrognathia are commonly encountered problems in pediatric plastic surgery. Mandibular distraction osteogenesis (MDO) is a relatively simple technique that allows for correction of the deformity with minimal morbidity. However, MDO can lead to a wide variety of complications. METHODS: The PubMed database was queried for all articles describing complications of MDO. Each article was then reviewed, and relevant data were extracted and compiled. Finally, several case reports are presented to illustrate poignant examples of complications. RESULTS: Complications of MDO include relapse (64.8% incidence), tooth injury (22.5%), hypertrophic scarring (15.6%), nerve injury (11.4%), infection (9.5%), inappropriate distraction vector (8.8%), device failure (7.9%), fusion error (2.4%), and temporomandibular joint injury (0.7%). CONCLUSIONS: Mandibular distraction osteogenesis can be associated with a wide variety of minor and major complications, but all complications can be avoided with careful planning and technique.

Regulation of yeast nutrient permease endocytosis by ATP-binding cassette transporters and a seven-transmembrane protein, RSB1.

Ceramide is produced by the condensation of a long chain base with a very long chain fatty acid. In Saccharomyces cerevisiae, one of the two major long chain bases is called phytosphingosine (PHS). PHS has been shown to cause toxicity in tryptophan auxotrophic strains of yeast because this bioactive ceramide precursor causes diversion of the high affinity tryptophan permease Tat2 to the vacuole rather than the plasma membrane. Loss of the integral membrane protein Rsb1 increased PHS sensitivity, which was suggested to be due to this protein acting as an ATP-dependent long chain base efflux protein. More recent experiments demonstrated that loss of the genes encoding the ATP-binding cassette transporter proteins Pdr5 and Yor1 elevated PHS tolerance. This increased resistance was suggested to be due to increased expression of RSB1. Here, we provide an alternative view of PHS resistance influenced by Rsb1 and Pdr5/Yor1. Rsb1 has a seven-transmembrane domain topology more consistent with that of a regulatory protein like a G-protein-coupled receptor rather than a transporter. Importantly, an rsb1Δ cell does not exhibit higher internal levels of PHS compared with isogenic wild-type cells. However, tryptophan transport is increased in pdr5Δ yor1 strains and reduced in rsb1Δ cells. Localization and vacuolar degradation of Tat2 are affected in these genetic backgrounds. Finally, internalization of FM4-64 dye suggests that loss of Pdr5 and Yor1 slows normal endocytic rates. Together, these data argue that Rsb1, Pdr5, and Yor1 regulate the endocytosis of Tat2 and likely other membrane transporter proteins.

Soft tissue to hard tissue advancement ratios for mandibular elongation using distraction osteogenesis in children.

Distraction osteogenesis is extensively used for the elongation of hypoplastic mandibles in children, yet the soft tissue profile response to this is not well understood. The pre- and posttreatment lateral cephalometric radiographs of 27 pediatric patients who underwent bilateral mandibular elongation using distraction osteogenesis were analyzed retrospectively to correlate horizontal soft tissue advancement with horizontal underlying bone advancement at B point and pogonion. Horizontal advancement (in millimeters) of bone and overlying soft tissue at these points was collected from the radiographs of each patient, and linear regression analysis was performed to determine the relationship of hard to soft tissue horizontal advancement at these points. A 1:0.90 mean ratio of bone to soft tissue advancement was observed at B point/labiomental sulcus and at pogonion/soft tissue pogonion (linear regression analysis demonstrated slopes [beta1 values] of 0.94 and 0.92, respectively). These ratios were consistent throughout the sample population and are highly predictive of the soft tissue response that can be anticipated. Magnitude of advancement, age, and sex of the patient had no effect on these ratios in our population. This study assists with our understanding of the soft tissue response that accompanies bony elongation during distraction osteogenesis which will allow us to more effectively treatment plan the orthodontic and surgical intervention that will optimize the patients' functional and esthetic outcome.

A yeast model system for functional analysis of the Niemann-Pick type C protein 1 homolog, Ncr1p.

Niemann-Pick disease type C (NP-C) is a progressive, ultimately fatal, autosomal recessive neurodegenerative disorder. The major biochemical hallmark of the disease is the endocytic accumulation of low-density lipoprotein-derived cholesterol. The majority of NP-C patients have mutations in the Niemann-Pick type C1 gene, NPC1. This study focuses on the Saccharomyces cerevisiae homolog of the human NPC1 protein encoded by the NCR1 gene. Ncr1p localizes to the vacuole, the yeast equivalent to the mammalian endosome-lysosome system. Here, we identify the first phenotype caused by deletion of NCR1 from the yeast genome, resistance to the ether lipid drug, edelfosine. Our results indicate that edelfosine has a cytotoxic, rather than cytostatic, effect on wildtype yeast cells. We exploit the edelfosine resistance phenotype to assess the function of yeast Ncr1 proteins carrying amino acid changes corresponding to human NPC1 patient mutations. We find that one of these amino acid changes severely compromises Ncr1p function as assessed using the edelfosine resistance assay. These findings establish S. cerevisiae as a model system that can be exploited to analyze the molecular consequences of patient mutations in NPC1 and provide the basis for future genetic studies using yeast.

Lem3p is essential for the uptake and potency of alkylphosphocholine drugs, edelfosine and miltefosine.

The alkylphosphocholine class of drugs, including edelfosine and miltefosine, has recently shown promise in the treatment of protozoal and fungal diseases, most notably, leishmaniasis. One of the major barriers to successful treatment of these infections is the development of drug resistance. To understand better the mechanisms underlying the development of drug resistance, we performed a combined mutant selection and screen in Saccharomyces cerevisiae, designed to identify genes that confer resistance to the alkylphosphocholine drugs by inhibiting their transport across the plasma membrane. Mutagenized cells were first selected for resistance to edelfosine, and the initial collection of mutants was screened a second time for defects in internalization of a short chain, fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD))-labeled phosphatidylcholine reporter. This approach identified mutations in a single gene, YNL323W/LEM3, that conferred resistance to alkylphosphocholine drugs and inhibited internalization of NBD-labeled phosphatidylcholine. Loss of YNL323W/LEM3 does not confer resistance to N-nitroquinilone N-oxide or ketoconazole and actually increases sensitivity to cycloheximide. The defect in internalization is specific to NBD-labeled phosphatidylcholine and phosphatidylethanolamine. Labeled phosphatidylserine is internalized at normal levels in lem3 strains. LEM3 is a member of an evolutionarily conserved family and has two homologues in S. cerevisiae. Single point mutations that produce resistance to alkylphosphocholine drugs and inhibition of NBD-labeled phosphatidylcholine internalization were identified in several highly conserved domains. These data demonstrate a requirement for Lem3p expression for normal phosphatidylcholine and alkylphosphocholine drug transport across the plasma membrane of yeast.

Rotation advancement of the midface by distraction osteogenesis.

A wide variety of disease processes produce alteration of midfacial skeletal growth, resulting in moderate-to-severe midface deficiency presenting as retrusion associated with Angle's class III malocclusion. Le Fort III osteotomies with advancement can provide an excellent tool for correction of this deformity. Recently, the corrective procedure of choice for advancement of midfacial segments has been distraction osteogenesis after osteotomy. Straight linear advancement is the most common choice for corrective movement of the midfacial segment, whether accomplished through acute surgical advancement or through the progressive distraction technique. Unfortunately, linear advancement can produce abnormal configurations, both at the nasal root and lateral orbits, regardless of the technique used. Enophthalmos, caused by orbital enlargement, may limit the advancement necessary to achieve class I occlusion. The authors have extended the utility of the Le Fort III procedure and have improved the final outcome by creating a controlled rotation advancement of the midfacial segment using distraction. The application of an existing internal distraction device is modified to control the movement of the midfacial segment in a rotation advancement path. Included in the series were 10 patients with severe midface retrusion secondary to multiple congenital syndromes, along with cleft lip and palate. The ages of the patients ranged from 6 to 14 years. An internal distraction system was used in all cases. Application of the distractor was substantially modified to simplify both fixation and removal and to produce controlled rotation advancement. The team orthodontist determined the final occlusal relationship. Percutaneous distractor drive rods were removed 4 to 6 weeks after active distraction to increase patient comfort. The distractors and all associated hardware were removed after 12 to 16 weeks of consolidation; follow-up periods ranged from 1 to 3 years. By using the modified distractor application to produce rotation advancement, the contour abnormalities at the nasal root and lateral orbit and the enophthalmos produced by linear advancement were eliminated. Significant improvement in facial contour and class I occlusion was obtained in all cases. Complications consisted of near exposure of the device in one patient. Stability has been excellent, with no relapse reported by the orthodontist. Rotational advancement of facial segments by distraction allows successful early intervention in patients with significant midface retrusion. The abnormal nasal root and lateral orbital configurations produced by direct linear advancement are avoided, and a stable and normalized facial configuration is produced.

Anatomic determinants of sleep-disordered breathing across the spectrum of clinical and nonclinical male subjects.

OBJECTIVES: We wished to determine the independent contribution of craniofacial dimensions of the upper airway to sleep-disordered breathing (SDB) in subjects who spanned the entire continuum of SDB. We also determined the interactive effects of body mass index (BMI) and age on the relationship between airway dimensions and SDB. DESIGN AND SUBJECTS: We studied 142 nonclinical male subjects in a working community population (average age, 47 years; average BMI, 29; average +/- SD apnea/hypopnea index [AHI], 20 +/- 20/h), and 62 patients with obstructive sleep apnea (average age, 47 years; average BMI, 32; average +/- SD AHI, 48 +/- 35/h. We determined the AHI from overnight polysomnography and the number of oxygen desaturations (> or = 2%) per hour of sleep. We used lateral facial cephalometric radiographs to measure 41 anatomic landmarks and 55 dimensions in the upper airway. SETTING: A university hospital and a sleep-disorders clinic. DATA ANALYSIS: We used stepwise regression analysis to determine the independent contributions of measured variables to SDB. MEASUREMENTS AND RESULTS: In the entire study population (n = 204), variations in BMI and six measures of craniofacial morphology accounted equally for one half of the total variance in AHI, and their interactive effects accounted for an additional 15%. Membership in the clinical or nonclinical group per se had no significant influence on these relationships. The single most important cephalometric variable in predicting AHI severity was the horizontal dimension of the maxilla (ie, porion vertical to supradentale [PV-A] distance). When the PV-A distance was relatively narrow (< 97 mm) the probability of having mild (AHI, 15 to 30/h) to severe (AHI > 30/h) SDB increased fivefold to sevenfold in nonobese subjects and threefold in obese subjects. Thus, in nonobese subjects (average BMI, 25 +/- 2) and in subjects with narrow upper airway dimensions, four cephalometric dimensions were the dominant predictors of AHI, accounting for 50% of the variance. However, in subjects with a large anteroposterior facial dimension, BMI was the major predictor of AHI and a BMI > 28 increased the probability of moderate-to-severe sleep apnea by approximately fivefold. Finally, the combination of cephalometric dimensions and BMI accounted for an increasing amount of the variance in AHI as the severity of AHI increased. CONCLUSIONS: Across the population spectrum of SDB, four cephalometric dimensions of the upper airway in combination with BMI accounted independently for up to two thirds of the variation in AHI; and the relative contribution of these two sets of determinants of AHI varied depending on airway size, obesity, and the amount of SDB.

NBD-labeled phosphatidylcholine enters the yeast vacuole via the pre-vacuolar compartment.

At low temperature, the short-chain fluorescent-labeled phospholipids, 1-myristoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) aminocaproyl]-phosphatidylcholine (M-C6-NBD-PC) and its phosphatidylethanolamine analog, M-C6-NBD-PE, are internalized by flip across the plasma membrane of S. cerevisiae and show similar enrichment in intracellular membranes including the mitochondria and nuclear envelope/ER. At higher temperatures (24-37 degrees C), or if low temperature internalization is followed by warming, M-C6-NBD-PC, but not M-C6-NBD-PE, is trafficked to the lumen of the vacuole. Sorting of M-C6-NBD-PC to the vacuole is blocked by energy-depletion and by null mutations in the VPS4 and VPS28 genes required for vesicular traffic from the pre-vacuolar compartment (PVC) to the vacuole. This sorting is not blocked by a temperature-sensitive mutation in SEC12, which inhibits ER to Golgi transport, a null mutation in VPS8, which inhibits Golgi to PVC transport, or temperature-sensitive and null mutations in END4, which inhibit endocytosis from the plasma membrane. Monomethylation or dimethylation of the primary amine head-group of M-C6-NBD-PE is sufficient for sorting to the yeast vacuole in both wild-type yeast and in strains defective in the phosphatidylethanolamine methylation pathway. These data indicate that methylation of M-C6-NBD-PE produces the crucial structural component required to sort these phospholipid analogues to the vacuole via the PVC.