Publications by authors named "Mark Schmitt"

24 Publications

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Peer-Developed Modules on Basic Biostatistics and Evidence-Based Medicine Principles for Undergraduate Medical Education.

MedEdPORTAL 2020 11 24;16:11026. Epub 2020 Nov 24.

Professor, Department of Physiological Sciences, Eastern Virginia Medical School.

Introduction: Evidence-based medicine (EBM) is pivotal in shaping patient care, yet it is challenging to incorporate into undergraduate medical education (UME) due to a lack of dedicated resources within the preclinical curriculum. To address this challenge, we used a peer-led approach to explain difficult concepts through language that students can understand at their shared level of understanding.

Methods: Four second-year medical students trained in EBM over 18 months by facilitating monthly journal clubs, ultimately leading to their involvement as peer-instructors. With input from a faculty expert, peer-instructors designed integrative PowerPoint modules and interactive problem sets on basic biostatistics and EBM principles. Assessment included formative quizzes with multiple attempts to achieve at least 80% to demonstrate mastery of core learning objectives. Afterwards, students were invited to provide feedback using a 5-point Likert scale survey.

Results: Of second-year students who participated, all 151 demonstrated 80% competency on each quiz. Eighty-seven (58%) students completed the survey on which, 77% agreed/strongly agreed that their level of understanding of EBM improved after the peer-led sessions, 76% agreed/strongly agreed that the sessions were more conducive to learning compared to traditional lectures, and 94% agreed/strongly agreed that the material covered was relevant to the USMLE Step 1.

Discussion: This peer-led approach has been rated as effective by learners, improving their ability to critically appraise and apply clinical evidence. To promote integration of EBM into UME, we have prepared modules, problem sets, quizzes, and an outline of the problem-solving sessions for universal adoption.
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http://dx.doi.org/10.15766/mep_2374-8265.11026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703476PMC
November 2020

Paneth Cells Respond to Inflammation and Contribute to Tissue Regeneration by Acquiring Stem-like Features through SCF/c-Kit Signaling.

Cell Rep 2018 08;24(9):2312-2328.e7

Department of Pathology, University Medical Center, Rotterdam, the Netherlands. Electronic address:

IBD syndromes such as Crohn's disease and ulcerative colitis result from the inflammation of specific intestinal segments. Although many studies have reported on the regenerative response of intestinal progenitor and stem cells to tissue injury, very little is known about the response of differentiated lineages to inflammatory cues. Here, we show that acute inflammation of the mouse small intestine is followed by a dramatic loss of Lgr5 stem cells. Instead, Paneth cells re-enter the cell cycle, lose their secretory expression signature, and acquire stem-like properties, thus contributing to the tissue regenerative response to inflammation. Stem cell factor secretion upon inflammation triggers signaling through the c-Kit receptor and a cascade of downstream events culminating in GSK3β inhibition and Wnt activation in Paneth cells. Hence, the plasticity of the intestinal epithelium in response to inflammation goes well beyond stem and progenitor cells and extends to the fully differentiated and post-mitotic Paneth cells.
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http://dx.doi.org/10.1016/j.celrep.2018.07.085DOI Listing
August 2018

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells.

J Vis Exp 2017 11 20(129). Epub 2017 Nov 20.

Department of Pathology, Erasmus MC Cancer Institute, Erasmus University Medical Center.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberkühn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate. Throughout the small intestine, Lgr5 stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5 stem cells through several key ligands as Wnt3, EGF, and Dll1. When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5 and Paneth cells. Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5 cells to associate and form organoids when co-cultured. This approach, here referred to as "organoid reconstitution assay" (ORA), allows the genetic and biochemical modification of Paneth or Lgr5 stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.
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http://dx.doi.org/10.3791/56329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755451PMC
November 2017

Modelling western dietary habits in the mouse: easier said than done.

Hepatobiliary Surg Nutr 2017 Apr;6(2):138-140

Department of Medicine and Cell Biology, Albert Einstein College of Medicine, New York, NY, USA.

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http://dx.doi.org/10.21037/hbsn.2017.01.20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411269PMC
April 2017

CD44v6-Peptide Functionalized Nanoparticles Selectively Bind to Metastatic Cancer Cells.

Adv Sci (Weinh) 2017 01 20;4(1):1600202. Epub 2016 Dec 20.

Institute of Toxicology and Genetics Karlsruhe Institute of Technology 76344 Karlsruhe Germany; Department of Applied Physical Chemistry University of Heidelberg 69120 Heidelberg Germany.

are fabricated in a facile and controllable way to selectively bind to CD44v6 positive tumor cells with highly efficient anticancer and antimetastatic properties. The reported modular synthesis and facile preparation makes this system highly potent for developing novel multifunctional nanocarriers for therapeutic and/or diagnostic anticancer applications.
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http://dx.doi.org/10.1002/advs.201600202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5238741PMC
January 2017

Low Fuel Convergence Path to Direct-Drive Fusion Ignition.

Phys Rev Lett 2016 Jun 24;116(25):255003. Epub 2016 Jun 24.

Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.

A new class of inertial fusion capsules is presented that combines multishell targets with laser direct drive at low intensity (2.8×10^{14}  W/cm^{2}) to achieve robust ignition. The targets consist of three concentric, heavy, metal shells, enclosing a volume of tens of μg of liquid deuterium-tritium fuel. Ignition is designed to occur well "upstream" from stagnation, with minimal pusher deceleration to mitigate interface Rayleigh-Taylor growth. Laser intensities below thresholds for laser plasma instability and cross beam energy transfer facilitate high hydrodynamic efficiency (∼10%).
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http://dx.doi.org/10.1103/PhysRevLett.116.255003DOI Listing
June 2016

CD44 regulates Wnt signaling at the level of LRP6.

Mol Cell Oncol 2015 Jul-Sep;2(3):e995046. Epub 2015 Jan 23.

Karlsruhe Institute of Technology; Institute of Toxicology and Genetics ; Postfach , Karlsruhe, Germany.

CD44 was recently identified as a positive feedback regulator of Wnt/β-catenin signaling. This regulation occurs at the level of low-density lipoprotein receptor-related protein 6 phosphorylation and membrane targeting. These findings broaden our understanding of the Wnt pathway activation process and open new perspectives for anti-CD44 therapies in diseases associated with Wnt induction, including colorectal cancer.
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http://dx.doi.org/10.4161/23723556.2014.995046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905321PMC
June 2016

Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer.

Cell Stem Cell 2016 07 9;19(1):38-51. Epub 2016 Jun 9.

Department of Pathology, Erasmus MC Cancer Institute, Rotterdam 3000CA, The Netherlands. Electronic address:

The intestinal stem cell niche provides cues that actively maintain gut homeostasis. Dysregulation of these cues may compromise intestinal regeneration upon tissue insult and/or promote tumor growth. Here, we identify secreted phospholipases A2 (sPLA2s) as stem cell niche factors with context-dependent functions in the digestive tract. We show that group IIA sPLA2, a known genetic modifier of mouse intestinal tumorigenesis, is expressed by Paneth cells in the small intestine, while group X sPLA2 is expressed by Paneth/goblet-like cells in the colon. During homeostasis, group IIA/X sPLA2s inhibit Wnt signaling through intracellular activation of Yap1. However, upon inflammation they are secreted into the intestinal lumen, where they promote prostaglandin synthesis and Wnt signaling. Genetic ablation of both sPLA2s improves recovery from inflammation but increases colon cancer susceptibility due to release of their homeostatic Wnt-inhibitory role. This "trade-off" effect suggests sPLA2s have important functions as genetic modifiers of inflammation and colon cancer.
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http://dx.doi.org/10.1016/j.stem.2016.05.023DOI Listing
July 2016

Occurrence of Meloidogyne fallax in North America, and Molecular Characterization of M. fallax and M. minor from U.S. Golf Course Greens.

Plant Dis 2013 Nov;97(11):1424-1430

School of Plant Sciences, The University of Arizona, Tucson 85721.

Several species of root-knot nematodes (Meloidogyne spp.) are known to have significant presence on turfgrass in golf course greens, particularly in the western United States. Nematodes isolated from a golf course in King County, WA were identified as Meloidogyne minor based on analysis of the large ribosomal subunit (LSU 28S D2-D3 expansion segment), the internal transcribed spacers 1 and 2 (ITS rDNA), the intergenic spacer region 2 (IGS2), and the nuclear protein-coding gene Hsp90. Sequence-characterized amplified region (SCAR) primers that were originally designed to be specific for M. fallax were found to cross-react with M. minor. A population from California was determined to be M. fallax based on juvenile tail morphology and analysis of the ribosomal markers and Hsp90, comprising the first report of this species in North America. Using trees based on Hsp90 genomic alignments, the phylogenetic relationships of these populations and known root-knot nematode species were congruent with previous trees based on ribosomal genes. Resolution of M. fallax and M. chitwoodi using Hsp90 was equivalent to species separation obtained with 28S or 18S rDNA alignments. The strengths and weaknesses of ribosomal and Hsp90 markers, and the use of SCAR polymerase chain reaction as diagnostic tools are discussed.
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http://dx.doi.org/10.1094/PDIS-03-13-0263-REDOI Listing
November 2013

Global identification of new substrates for the yeast endoribonuclease, RNase mitochondrial RNA processing (MRP).

J Biol Chem 2012 Oct 12;287(44):37089-97. Epub 2012 Sep 12.

Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.

RNase mitochondrial RNA processing (MRP) is an essential, evolutionarily conserved endoribonuclease composed of 10 different protein subunits and a single RNA. RNase MRP has established roles in multiple pathways including ribosome biogenesis, cell cycle regulation, and mitochondrial DNA replication. Although each of these functions is important to cell growth, additional functions may exist given the essential nature of the complex. To identify novel RNase MRP substrates, we utilized RNA immunoprecipitation and microarray chip analysis to identify RNA that physically associates with RNase MRP. We identified several new potential substrates for RNase MRP including a cell cycle-regulated transcript, CTS1; the yeast homolog of the mammalian p27(Kip1), SIC1; and the U2 RNA component of the spliceosome. In addition, we found RNase MRP to be involved in the regulation of the Ty1 transposon RNA. These results reinforce and broaden the role of RNase MRP in cell cycle regulation and help to identify new roles of this endoribonuclease.
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http://dx.doi.org/10.1074/jbc.M112.389023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3481309PMC
October 2012

Root-Knot Nematodes in Golf Course Greens of the Western United States.

Plant Dis 2012 May;96(5):635-647

California Department of Food and Agriculture, Sacramento 95832.

A survey of 238 golf courses in 10 states of the western United States found root-knot nematodes (Meloidogyne spp.) in 60% of the putting greens sampled. Sequence and phylogenetic analyses of 18S rRNA, D2-D3 of 28S rRNA, internal transcribed spacer-rRNA, and mitochondrial DNA gene sequences were used to identify specimens from 110 golf courses. The most common species, Meloidogyne naasi, was found in 58 golf courses distributed from Southern California to Washington in the coastal or cooler areas of those states. In the warmer regions of the Southwest, M. marylandi was recovered from 38 golf courses and M. graminis from 11 golf courses. This constitutes the first report of M. marylandi in Arizona, California, Hawaii, Nevada, and Utah, and the first report of M. graminis in Arizona, Hawaii, and Nevada. Two golf courses in Washington were infested with M. minor, the first record of this nematode in the Western Hemisphere. Columbia root-knot nematode, M. chitwoodi, was found in a single golf course in California. Polymerase chain reaction restriction fragment length polymorphism of the intergenic region between the cytochrome oxidase and 16S rRNA genes in the mitochondrial genome with restriction enzyme SspI was able to distinguish populations of M. graminis from M. marylandi, providing a fast and inexpensive method for future diagnosis of these nematodes from turf.
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http://dx.doi.org/10.1094/PDIS-09-11-0808DOI Listing
May 2012

Transcriptome analysis of a barley breeding program examines gene expression diversity and reveals target genes for malting quality improvement.

BMC Genomics 2010 Nov 23;11:653. Epub 2010 Nov 23.

Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, 55108, USA.

Background: Advanced cycle breeding utilizes crosses among elite lines and is a successful method to develop new inbreds. However, it results in a reduction in genetic diversity within the breeding population. The development of malting barley varieties requires the adherence to a narrow malting quality profile and thus the use of advanced cycle breeding strategies. Although attention has been focused on diversity in gene expression and its association with genetic diversity, there are no studies performed in a single breeding program examining the implications that consecutive cycles of breeding have on gene expression variation and identifying the variability still available for future improvement.

Results: Fifteen lines representing the historically important six-rowed malting barley breeding program of the University of Minnesota were genotyped with 1,524 SNPs, phenotypically examined for six malting quality traits, and analyzed for transcript accumulation during germination using the Barley1 GeneChip array. Significant correlation was detected between genetic and transcript-level variation. We observed a reduction in both genetic and gene expression diversity through the breeding process, although the expression of many genes have not been fixed. A high number of quality-related genes whose expression was fixed during the breeding process was identified, indicating that much of the diversity reduction was associated with the improvement of the complex phenotype "malting quality", the main goal of the University of Minnesota breeding program. We also identified 49 differentially expressed genes between the most recent lines of the program that were correlated with one or more of the six primary malting quality traits. These genes constitute potential targets for the improvement of malting quality within the breeding program.

Conclusions: The present study shows the repercussion of advanced cycle breeding on gene expression diversity within an important barley breeding program. A reduction in gene expression diversity was detected, although there is diversity still present after forty years of breeding that can exploited for future crop improvement. In addition, the identification of candidate genes for enhancing malting quality may be used to optimize the selection of targets for further improvements in this economically important phenotype.
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http://dx.doi.org/10.1186/1471-2164-11-653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091773PMC
November 2010

Role of RNase MRP in viral RNA degradation and RNA recombination.

J Virol 2011 Jan 20;85(1):243-53. Epub 2010 Oct 20.

Department of Plant Pathology, University of Kentucky, 201F Plant Science Building, Lexington, KY 40546-0091, USA.

RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.
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http://dx.doi.org/10.1128/JVI.01749-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014185PMC
January 2011

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components.

RNA 2010 Mar 19;16(3):529-37. Epub 2010 Jan 19.

Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.
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http://dx.doi.org/10.1261/rna.1893710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2822918PMC
March 2010

Distribution, Biology and Pathology of Anguina pacificae.

J Nematol 2008 Sep;40(3):226-39

Department of Plant Sciences, University of Arizona, Tucson, AZ85721, Northern California Golf Association, P.O. Box NCGA, Pebble Beach, CA 93953.

Anguina pacificae is distributed along a narrow strip on the Pacific coast of Northern California where it forms galls on the shoots of Poa annua and causes significant damage to golf course greens. Methods were developed for the continuous propagation of A. pacificae on P. annua in growth chambers, and they were used to examine the life cycle and host-parasite relationships of the nematode. At a mean temperature of 20 degrees C (22 degrees C day/18 degrees C night) the life cycle was completed in as little as 32 days (inoculation to second-generation J2). The first molt occurred in the egg. Infective J2 hatched from the eggs and penetrated the shoot near the crown of the plant where a cavity was formed 200 to 300 mum below the shoot apex. A gall around the cavity was visible 12 days after inoculation (DAI), and the cavity and gall continued to enlarge until second-generation J2 began to hatch. Three additional molts occurred in the cavity of the developing gall 14 to 24 DAI. Sexes could be distinguished 15 DAI. Egg production began 26 DAI and continued for 10 to 15 days. Eggs commenced hatching inside the gall 42 DAI, when the adults began to die and decompose. By 57 DAI, the gall had reached its maximum diameter, and the cavity was filled entirely with second-generation J2 that remained in the gall until they were liberated when the gall decomposed. J2 in galls survived desiccation over silica gel for 14 months at 14 degrees C and were active and infective when rehydrated.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664668PMC
September 2008

Footprinting analysis demonstrates extensive similarity between eukaryotic RNase P and RNase MRP holoenzymes.

RNA 2008 Aug 25;14(8):1558-67. Epub 2008 Jun 25.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

Eukaryotic ribonuclease (RNase) P and RNase MRP are evolutionary related RNA-based enzymes involved in metabolism of various RNA molecules, including tRNA and rRNA. In contrast to the closely related eubacterial RNase P, which is comprised of an RNA component and a single small protein, these enzymes contain multiple protein components. Here we report the results of footprinting studies performed on purified Saccharomyces cerevisiae RNase MRP and RNase P holoenzymes. The results identify regions of the RNA components affected by the protein moiety, suggest a role of the proteins in stabilization of the RNA fold, and point to substantial similarities between the two evolutionary related RNA-based enzymes.
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http://dx.doi.org/10.1261/rna.1106408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2491465PMC
August 2008

Specific binding of a Pop6/Pop7 heterodimer to the P3 stem of the yeast RNase MRP and RNase P RNAs.

RNA 2007 Oct 23;13(10):1648-55. Epub 2007 Aug 23.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

Pop6 and Pop7 are protein subunits of Saccharomyces cerevisiae RNase MRP and RNase P. Here we show that bacterially expressed Pop6 and Pop7 form a soluble heterodimer that binds the RNA components of both RNase MRP and RNase P. Footprint analysis of the interaction between the Pop6/7 heterodimer and the RNase MRP RNA, combined with gel mobility assays, demonstrates that the Pop6/7 complex binds to a conserved region of the P3 domain. Binding of these proteins to the MRP RNA leads to local rearrangement in the structure of the P3 loop and suggests that direct interaction of the Pop6/7 complex with the P3 domain of the RNA components of RNases MRP and P may mediate binding of other protein components. These results suggest a role for a key element in the RNase MRP and RNase P RNAs in protein binding, and demonstrate the feasibility of directly studying RNA-protein interactions in the eukaryotic RNases MRP and P complexes.
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http://dx.doi.org/10.1261/rna.654407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1986809PMC
October 2007

Microscopy reveals disease control through novel effects on fungal development: a case study with an early-generation benzophenone fungicide.

Pest Manag Sci 2006 May;62(5):383-92

USDA-ARS, Cereal Crops Research Unit, 501 Walnut Street, Madison, WI 53726, USA.

The benzophenones are a new class of agricultural fungicides that demonstrate protectant, curative and eradicative/antisporulant activity against powdery mildews. The chemistry is represented in the marketplace by the fungicide metrafenone, recently introduced by BASF and discussed in the following paper. The benzophenones show no evidence of acting by previously identified biochemical mechanisms, nor do they show cross-resistance with existing fungicides. The value of microscopy in elucidating fungicide mode of action is demonstrated through identification of the effects of an early benzophenone, eBZO, on mildew development. eBZO caused profound alterations in the morphology of powdery mildews of both monocotyledons and dicotyledons, affecting multiple stages of fungal development, including spore germination, appressorial formation, penetration, surface hyphal morphology and sporogenesis. Identification of analogous effects of eBZO on sporulation in the model organism Aspergillus nidulans (Eidam) Winter provides a unique opportunity to elucidate important morphogenetic regulatory sites in the economically important obligate pathogens, the powdery mildews. Benzophenones provide a further example of the benefits of whole-organism testing in the search for novel fungicide modes of action.
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http://dx.doi.org/10.1002/ps.1177DOI Listing
May 2006

A specialized processing body that is temporally and asymmetrically regulated during the cell cycle in Saccharomyces cerevisiae.

J Cell Biol 2006 Apr 3;173(1):35-45. Epub 2006 Apr 3.

Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.

RNase mitochondrial RNA processing (MRP) is an essential ribonucleoprotein endoribonuclease that functions in the degradation of specific mRNAs involved in cell cycle regulation. We have investigated where this processing event occurs and how it is regulated. As expected, results demonstrate that RNase MRP is predominantly localized in the nucleolus, where it processes ribosomal RNAs. However, after the initiation of mitosis, RNase MRP localizes throughout the entire nucleus and in a single discrete cytoplasmic spot that persists until the completion of telophase. Furthermore, this spot was asymmetrically found in daughter cells, where the RNase MRP substrate, CLB2 mRNA, localizes. Both the mitotic exit network and fourteen early anaphase release pathways are nonessential but important for the temporal changes in localization. Asymmetric localization was found to be dependent on the locasome. The evidence suggests that these spots are specialized processing bodies for the degradation of transcripts that are cell cycle regulated and daughter cell localized. We have called these TAM bodies for temporal asymmetric MRP bodies.
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http://dx.doi.org/10.1083/jcb.200512025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2063784PMC
April 2006

Consequences of mutations in the non-coding RMRP RNA in cartilage-hair hypoplasia.

Hum Mol Genet 2005 Dec 27;14(23):3723-40. Epub 2005 Oct 27.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Cartilage-hair hypoplasia (CHH), also known as metaphyseal chondrodysplasia McKusick type (OMIM no. 250250), is an autosomal recessive, multi-systemic disease characterized by disproportionate short stature, fine and sparse hair, deficient cellular immunity and a predisposition to malignancy. It is caused by mutations in RMRP, the RNA component of the ribonucleoprotein complex RNase MRP, and, thus, CHH represents one of few Mendelian disorders caused by mutations in a nuclear encoded, non-coding RNA. While studies in yeast indicate that RMRP contributes to diverse cellular functions, the pathogenesis of the human condition is unknown. Studies of our CHH patient cohort revealed mutations in both the promoter and the transcribed region of RMRP. While mutations in the promoter abolished transcription in vitro, RMRP RNA levels in patients with transcribed mutations were also decreased suggesting an unstable RNA. RMRP mutations introduced into the yeast ortholog, NME1, exhibited normal mitochondrial function, chromosomal segregation and cell cycle progression, while a CHH fibroblast cell line exhibited normal mitochondrial content. However, the most commonly found mutation in CHH patients, 70A>G, caused an alteration in ribosomal processing by altering the ratio of the short versus the long form of the 5.8S rRNA in yeast. Transcriptional profiling of CHH patient RNAs showed upregulation of several cytokines and cell cycle regulatory genes, one of which has been implicated in chondrocyte hypertrophy. These data suggest that alteration of ribosomal processing in CHH is associated with altered cytokine signalling and cell cycle progression in terminally differentiating cells in the lymphocytic and chondrocytic cell lineages.
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http://dx.doi.org/10.1093/hmg/ddi403DOI Listing
December 2005

Severely incapacitating mutations in patients with extreme short stature identify RNA-processing endoribonuclease RMRP as an essential cell growth regulator.

Am J Hum Genet 2005 Nov 29;77(5):795-806. Epub 2005 Sep 29.

Institute of Human Genetics, University of Erlangen-Nurnberg, Erlangen, Germany.

The growth of an individual is deeply influenced by the regulation of cell growth and division, both of which also contribute to a wide variety of pathological conditions, including cancer, diabetes, and inflammation. To identify a major regulator of human growth, we performed positional cloning in an autosomal recessive type of profound short stature, anauxetic dysplasia. Homozygosity mapping led to the identification of novel mutations in the RMRP gene, which was previously known to cause two milder types of short stature with susceptibility to cancer, cartilage hair hypoplasia, and metaphyseal dysplasia without hypotrichosis. We show that different RMRP gene mutations lead to decreased cell growth by impairing ribosomal assembly and by altering cyclin-dependent cell cycle regulation. Clinical heterogeneity is explained by a correlation between the level and type of functional impairment in vitro and the severity of short stature or predisposition to cancer. Whereas the cartilage hair hypoplasia founder mutation affects both pathways intermediately, anauxetic dysplasia mutations do not affect B-cyclin messenger RNA (mRNA) levels but do severely incapacitate ribosomal assembly via defective endonucleolytic cleavage. Anauxetic dysplasia mutations thus lead to poor processing of ribosomal RNA while allowing normal mRNA processing and, therefore, genetically separate the different functions of RNase MRP.
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http://dx.doi.org/10.1086/497708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1271388PMC
November 2005

Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component.

J Biol Chem 2005 Mar 6;280(12):11352-60. Epub 2005 Jan 6.

Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.

In the yeast Saccharomyces cerevisiae, RNase mitochondrial RNA processing (MRP) is an essential endoribonuclease that consists of one RNA component and at least nine protein components. Characterization of the complex is complicated by the fact that eight of the known protein components are shared with a related endoribonuclease, RNase P. To fully characterize the RNase MRP complex, we purified it to apparent homogeneity in a highly active state using tandem affinity purification. In addition to the nine known protein components, both Rpr2 and a protein encoded by the essential gene YLR145w were present in our preparations of RNase MRP. Precipitation of a tagged version of Ylr145w brought with it the RNase MRP RNA, but not the RNase P RNA. A temperature-sensitive ylr145w mutant was generated and found to exhibit a rRNA processing defect identical to that seen in other RNase MRP mutants, whereas no defect in tRNA processing was observed. Homologues of the Ylr145w protein were found in most yeasts, fungi, and Arabidopsis. Based on this evidence, we propose that YLR145w encodes a novel protein component of RNase MRP, but not RNase P. We recommend that this gene be designated RMP1, for RNase MRP protein 1.
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http://dx.doi.org/10.1074/jbc.M409568200DOI Listing
March 2005

RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation.

Mol Cell Biol 2004 Feb;24(3):945-53

Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.

RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC321458PMC
http://dx.doi.org/10.1128/mcb.24.3.945-953.2004DOI Listing
February 2004

The Saccharomyces cerevisiae RNase mitochondrial RNA processing is critical for cell cycle progression at the end of mitosis.

Genetics 2002 Jul;161(3):1029-42

Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 50 E Adams Street, Syracuse, NY 13210, USA.

We have identified a cell cycle delay in Saccharomyces cerevisiae RNase MRP mutants. Mutants delay with large budded cells, dumbbell-shaped nuclei, and extended spindles characteristic of "exit from mitosis" mutants. In accord with this, a RNase MRP mutation can be suppressed by overexpressing the polo-like kinase CDC5 or by deleting the B-type cyclin CLB1, without restoring the MRP-dependent rRNA-processing step. In addition, we identified a series of genetic interactions between RNase MRP mutations and mutations in CDC5, CDC14, CDC15, CLB2, and CLB5. As in most "exit from mitosis" mutants, levels of the Clb2 cyclin were increased. The buildup of Clb2 protein is not the result of a defect in the release of the Cdc14 phosphatase from the nucleolus, but rather the result of an increase in CLB2 mRNA levels. These results indicate a clear role of RNase MRP in cell cycle progression at the end of mitosis. Conservation of this function in humans may explain many of the pleiotropic phenotypes of cartilage hair hypoplasia.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462176PMC
July 2002