Biochemistry, Biophysics, and Structural Biology Commons^™

Development Of Novel Biomarkers In Cancer: Detection Of Circulating Mir-141 As A Potential Prognostic Marker For Prostate Cancer, Jason Cadaoas Gonzales

UNLV Theses, Dissertations, Professional Papers, and Capstones

Prostate cancer (CAP) is the most common epithelial malignancy and the second leading cause of cancer deaths in American men. The identification of predictive and prognostic biomarkers in CAP patients is critical for improving clinical outcomes. Although the measurement of prostate-specific antigen (PSA) and radiographic studies are clinically approved to predict response to therapy, these tests can oftentimes prove to be inadequate in certain patients. Thus, it is important to discover new biomarkers to improve chances of survivability. We and others have shown that longitudinal measurements of circulating tumor cells (CTC) and lactate dehydrogenase (LDH) may aid in predicting response …

The Mir-15/107 Group Of Microrna Genes: Evolutionary Biology, Cellular Functions, And Roles In Human Diseases, John R. Finnerty, Wang-Xia Wang, Sébastien S. Hébert, Bernard R. Wilfred, Guogen Mao, Peter T. Nelson

Pathology and Laboratory Medicine Faculty Publications

The miR-15/107 group of microRNA (miRNA) gene is increasingly appreciated to serve key functions in humans. These miRNAs regulate gene expression involved in cell division, metabolism, stress response, and angiogenesis in vertebrate species. The miR-15/107 group has also been implicated in human cancers, cardiovascular disease and neurodegenerative disease, including Alzheimer's disease. Here we provide an overview of the following: (1) the evolution of miR-15/107 group member genes; (2) the expression levels of miRNAs in mammalian tissues; (3) evidence for overlapping gene-regulatory functions by different miRNAs; (4) the normal biochemical pathways regulated by miR-15/107 group miRNAs; and (5) the roles played …

Notch1 Functions As A Tumor Suppressor In A Model Of K-Ras–Induced Pancreatic Ductal Adenocarcinoma, Linda Hanlon, Jacqueline L Avila, Renée M Demarest, Scott Troutman, Megan Allen, Francesca Ratti, Anil K Rustgi, Ben Z Stanger, Fred Radtke, Volkan Adsay, Fenella Long, Anthony J Capobianco, Joseph L Kissil

Rowan-Virtua School of Osteopathic Medicine Departmental Research

K-ras is the most commonly mutated oncogene in pancreatic cancer and its activation in murine models is sufficient to recapitulate the spectrum of lesions seen in human pancreatic ductal adenocarcinoma (PDAC). Recent studies suggest that Notch receptor signaling becomes reactivated in a subset of PDACs, leading to the hypothesis that Notch1 functions as an oncogene in this setting. To determine whether Notch1 is required for K-ras-induced tumorigenesis, we used a mouse model in which an oncogenic allele of K-ras is activated and Notch1 is deleted simultaneously in the pancreas. Unexpectedly, the loss of Notch1 in this model resulted in increased …

Ricin B Chain-Insulin Fusion Protein Immunomodulation Of Type 1 Diabetes, James Edward Carter Iii

Loma Linda University Electronic Theses, Dissertations & Projects

Type 1 diabetes mellitus (T1D) is a debilitating chronic inflammatory disease of the insulin-producing pancreatic islet β-cells that results from a combination of genetic and environmental factors. Attempts to suppress Th1-mediated autoimmune diseases such as T1D by mucosal delivery of autoantigens for immunotolerization have yielded only partial success. Attainment of satisfactory levels of sustained immunological tolerance remains to be accomplished. To restore self-tolerance requires delivery of sufficient amounts of autoantigen to stimulate regulatory T helper cells that function to survey the gut and induce tolerance to consumed antigens such as food. Oral delivery of autoantigens has previously been shown to …

Aspergillus Fumigatus Stimulates The Nlrp3 Inflammasome Through A Pathway Requiring Ros Production And The Syk Tyrosine Kinase, Najwane Saïd-Sadier, Eduardo V. Padilla, Gordon Langsley, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Invasive aspergillosis (IA) is a life-threatening disease that occurs in immunodepressed patients when infected with Aspergillus fumigatus. This fungus is the second most-common causative agent of fungal disease after Candida albicans. Nevertheless, much remains to be learned about the mechanisms by which A. fulmigatus activates the innate immune system. We investigated the inflammatory response to conidia and hyphae of A. fumigatus and specifically, their capacity to trigger activation of an inflammasome. Our results show that in contrast to conidia, hyphal fragments induce NLRP3 inflammasome assembly, caspase-1 activation and IL-1β release from a human monocyte cell line. The ability of Aspergillus …

Efficient Activation Of Reconstructed Rat Embryos By Cyclin-Dependent Kinase Inhibitors, Robin L. Webb, Kirk A. Findlay, Michael A. Green, Tina L. Beckett, M. Paul Murphy

Molecular and Cellular Biochemistry Faculty Publications

Background

Over the last decade a number of species, from farm animals to rodents, have been cloned using somatic cell nuclear transfer technology (SCNT). This technique has the potential to revolutionize the way that genetically modified animals are made. In its current state, the process of SCNT is very inefficient (<5% success rate), with several technical and biological hurdles hindering development. Yet, SCNT provides investigators with powerful advantages over other approaches, such as allowing for prescreening for the desired level of transgene expression and eliminating the excess production of undesirable wild-type animals. The rat plays a significant role in biomedical research, but SCNT has been problematic for this species. In this study, we address one aspect of the problem by evaluating methods of activation in artificially constructed rat embryos.

Principal Findings

We demonstrate that treatment with a calcium ionophore (ionomycin) combined with a variety of cyclin-dependent kinase inhibitors is an effective way to activate rat embryos. This is in contrast to methods developed for the mouse embryo, which tolerates much less specific chemical treatments. Methods developed to activate …

Role Of All Of The Prrsv Glycoproteins In Protective Immune Response, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions amongst each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by co-immunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that strong interaction exists between GP4 and GP5 proteins, although weak interactions …

Maternal Consumption Of Canola Oil Suppressed Mammary Gland Tumorigenesis In C3(1) Tag Mice Offspring, Gabriela Ion, Juliana A. Akinsete, W. Elaine Hardman

Biochemistry and Microbiology

Background: Maternal consumption of a diet high in omega 6 polyunsaturated fats (n-6 PUFA) has been shown to increase risk whereas a diet high in omega 3 polyunsaturated fats (n-3 PUFA) from fish oil has been shown to decrease risk for mammary gland cancer in female offspring of rats. The aim of this study was to determine whether increasing n-3 PUFA and reducing n-6 PUFA by using canola oil instead of corn oil in the maternal diet might reduce the risk for breast cancer in female offspring.

Methods: Female SV 129 mice were divided into two groups and placed on …

Cell-Type Specific Distribution Of Chloride Transporters In The Rat Suprachiasmatic Nucleus, Michael A. Belenky, Patricia J. Sollars, David B. Mount, Seth L. Alper, Yosef Yarom, Gary E. Pickard

School of Veterinary and Biomedical Sciences: Faculty Publications

The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use γ-aminobutyric acid (GABA) as a neurotransmitter, the principal inhibitory neurotransmitter in the adult central nervous system. Acting via the ionotropic GABAA receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl− influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function …

Porcine Reproductive And Respiratory Syndrome Virus Nonstructural Protein 1Β Modulates Host Innate Immune Response By Antagonizing Irf3 Activation, Lalit Beura, Saumendra Sarkar, Byungjoon Kwon, Sakthivel Subramaniam, Clinton J. Jones, Asit K. Pattnaik, Fernando A. Osorio

School of Veterinary and Biomedical Sciences: Faculty Publications

Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-β) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, …

The Minor Envelope Glycoproteins Gp2a And Gp4 Of Porcine Reproductive And Respiratory Syndrome Virus Interact With The Receptor Cd163, Phani B. Das, Phat X. Dinh, Israrul H. Ansari, Marcelo De Lima, Fernando A. Osorio, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions with each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by coimmunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that a strong interaction exists between the GP4 and GP5 proteins, although …

Prospects And Pits On The Path Of Biomimetics: The Case Of Tooth Enamel, Vuk Uskoković

Pharmacy Faculty Articles and Research

This review presents a discourse on challenges in understanding and imitating the process of amelogenesis in vitro on the molecular scale. In light of the analysis of imitation of the growth of dental enamel, it also impends on the prospects and potential drawbacks of the biomimetic approach in general. As the formation of enamel proceeds with the protein matrix guiding the crystal growth, while at the same time conducting its own degradation and removal, it is argued that three aspects of amelogenesis need to be induced in parallel: a) crystal growth; b) protein assembly; c) proteolytic degradation. A particular emphasis …

Ligand-Protein Interactions Of Inhibitors With Dengue Virus Type 2 Serine Protease : A Structural Study., Rozana Othman

Student Works (2010-2019)

Dengue is a serious infectious disease that is endemic in over 100 countries. There has been an estimate of 50 million infection per year globally, with more than 2.5 billion people are at risk for epidemic transmission. Two principal illnesses associated with dengue are Dengue Fever (DF) and Dengue Haemorrhagic Fever (DHF). To date, there is no licensed vaccine or therapeutic drug available for these illnesses, although there have been reports of some vaccine candidates in clinical trials. Treatments have only been supportive thus far. This thesis describes part of our ongoing effort to search for a lead therapeutic agent …

The Interictal State In Epilepsy And Behavior, Daniel Tice Barkmeier

Wayne State University Dissertations

Epilepsy is one of the most common neurological diseases, affecting up to 1% of the world population. Epilepsy remains poorly understood and there are currently no medications to cure it. Patients with epilepsy have both seizures as well as another type of abnormal activity between seizures, known as interictal spikes. Interictal spikes have thus far been poorly researched, yet growing evidence supports an important role for them in epilepsy. In this project, we first show the high variability between reviewers in marking interictal spikes on intracranial EEG, and then develop and test an automated detection method to solve this problem. …

Induction Of Interferon And Interferon Signaling Pathways By Replication Of Defective Interfering Particle Rna In Cells Constitutively Expressing Vesicular Stomatitis Virus Replication Proteins, Debasis Panda, Phat X. Dinh, Lalit Beura, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

We show here that replication of defective interfering (DI) particle RNA in HEK293 cells stably expressing vesicular stomatitis virus (VSV) replication proteins potently activates interferon (IFN) and IFN signaling pathways through upregulation of IFN- promoter, IFN-stimulated response element (ISRE) promoter, and NF-κB promoter activities. Replication of DI particle RNA, not mere expression of the viral replication proteins, was found to be critical for induction of IFN and IFN signaling. The stable cells supporting replication of DI RNA described in this report will be useful in further examining the innate immune signaling pathways and the host cell functions in viral genome …

Towards An Understanding Of The Herpes Simplex Virus Type 1 Latency-Reactivation Cycle, Guey-Chuen Perng, Clinton Jones

School of Veterinary and Biomedical Sciences: Faculty Publications

Infection by herpes simplex virus type 1 (HSV-1) can cause clinical symptoms in the peripheral and central nervous system. Recurrent ocular shedding can lead to corneal scarring and vision loss making HSV-1 a leading cause of corneal blindness due to an infectious agent. The primary site of HSV-1 latency is sensory neurons within trigeminal ganglia. Periodically, reactivation from latency occurs resulting in virus transmission and recurrent disease. During latency, the latency-associated transcript (LAT) is abundantly expressed. LAT expression is important for the latency-reactivation cycle in animal models, in part, because it inhibits apoptosis, viral gene expression, and productive infection. A …

Method For Identification Of Virulence Determinants, Raul G. Barletta, Beth Harris

School of Veterinary and Biomedical Sciences: Faculty Publications

Disclosed are methods for the determination of virulence determinants in bacteria and in particular bacteria of the genus Mycobacterium. Also disclosed are compositions and methods for stimulating an immune response in an animal using bacteria and virulence determinants identified by the methods of the present invention.

Biochemical Characterization Of Human Mismatch Recognition Proteins Mutsα And Mutsβ, Lei Tian

University of Kentucky Doctoral Dissertations

The integrity of an organism's genome depends on the fidelity of DNA replication and the efficiency of DNA repair. The DNA mismatch repair (MMR) system, which is highly conserved from prokaryotes to eukaryotes, plays an important role in maintaining genome stability by correcting base-base mismatches and insertion/deletion (ID) mispairs generated during DNA replication and other DNA transactions. Mismatch recognition is a critical step in MMR. Two mismatch recognition proteins, MutSα (MSH2-MSH6 heterodimer) and MutSβ (MSH2-MSH3 heterodimer), have been identified in eukaryotic cells. MutSα and MutSβ have partially overlapping functions, with MutSα recognizing primarily base-base mismatches and 1-2 nt ID mispairs …

Explorations In Homeoviscous Adaptation And Mass Spectral Analysis Of Membrane Lipids, Michael Douglas Timmons

University of Kentucky Doctoral Dissertations

The focus of this dissertation is centered on the mass spectral analysis of lipids and changes occurring in keeping with the concept of homeoviscous adaptation [1]. Homeoviscous adaptation is the process of modification of membrane lipids in response to environmental stimuli [1]. Dissertation investigations applied this concept to prokaryotic and eukaryotic organisms, and expanded the perception of environmental factors from exogenous organic solvents to intracellular environment.

The field of lipidomics deals with the analysis of phospholipid and fatty acid components of membranes the changes that occur due to environmental stimuli and their biological significance [2-6]. The high sensitivity of mass …

The Globin Gene Family Of The Cephalochordate Amphioxus: Implications For Chordate Globin Evolution, Bettina Ebner, Georgia Panopoulou, Serge N. Vinogradov, Laurent Kiger, Michael C. Marden, Thorsten Burmester, Thomas Hankeln

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire.

Results

We show that B. floridae harbors at least fifteen paralogous globin genes, …

The Danger Signal Adenosine Induces Persistence Of Chlamydial Infection Through Stimulation Of A2b Receptors, Matthew A. Pettengill, Verissa W. Lam, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the invading pathogen, leading also to local tissue damage and hypoxia. However, the effects of key aspects of damaged inflamed tissues and hypoxia on continued infection with intracellular bacteria remain unknown. We find that development of Chlamydia trachomatis is reversibly retarded by prolonged exposure of infected cells to extracellular adenosine, a hallmark of hypoxia and advanced inflammation. In epithelial cells, this effect was mediated by the A2b adenosine receptor, unique in the adenosine receptor …

Inactivation Of The Fliy Gene Encoding A Flagellar Motor Switch Protein Attenuates Mobility And Virulence Of Leptospira Interrogans Strain Lai, Sumei Liao, Ai-Hua Sun, David M. Ojcius, Senlin Wu, Jinfang Zhao, Jie Yan

All Dugoni School of Dentistry Faculty Articles

Background: Pathogenic Leptospira species cause leptospirosis, a zoonotic disease of global importance. The spirochete displays active rotative mobility which may contribute to invasion and diffusion of the pathogen in hosts. FliY is a flagellar motor switch protein that controls flagellar motor direction in other microbes, but its role in Leptospira, and paricularly in pathogenicity remains unknown.

Results: A suicide plasmid for the fliY gene of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai that was disrupted by inserting the ampicillin resistance gene (bla) was constructed, and the inactivation of fliY gene in a mutant (fliY-) was confirmed by PCR and …

Expression And Function Of Hypoxia Inducible Factor-1 Alpha In Human Melanoma Under Non-Hypoxic Conditions, Caroline N. Mills, Sandeep S. Joshi, Richard M. Niles

Biochemistry and Microbiology

Background

Hypoxia inducible factor-1 alpha (HIF-1α) protein is rapidly degraded under normoxic conditions. When oxygen tensions fall HIF-1α protein stabilizes and transactivates genes involved in adaptation to hypoxic conditions. We have examined the normoxic expression of HIF-1α RNA and protein in normal human melanocytes and a series of human melanoma cell lines isolated from radial growth phase (RGP), vertical growth phase (VGP) and metastatic (MET) melanomas.

Results

HIF-1α mRNA and protein was increased in RGP vs melanocytes, VGP vs RGP and MET vs VGP melanoma cell lines. We also detected expression of a HIF-1α mRNA splice variant that lacks part …

Methods And Compositions For Vaccnation Of Animals With Prrsv Antigens With Improved Immunogenicity, Israrul H. Ansari, Fernando A. Osorio, Asit K. Pattnaik,

School of Veterinary and Biomedical Sciences: Faculty Publications

Pigs challenged with hypoglycosylated variants of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) major surface protein GP5 exhibited increased production of PRRSV-neutralizing antibodies relative to the levels of neutralizing antibodies produced by pigs immunized with wild type (wt) or glycosylated GP5. This invention provides for methods of obtaining improved immune responses in pigs to PRRSV, compositions useful for obtaining the improved immune responses as well as isolated polynucleotides that encode hypoglycosylated variants of PRRSV major surface protein GP5.

Inflammasome-Dependent Caspase-1 Activation In Cervical Epithelial Cells Stimulates Growth Of The Intracellular Pathogen Chlamydia Trachomatis, Ali A. Abdul-Sater, Evonne Koo, Georg Hacker, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K+ through glibenclamide-sensitive K+ channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive …

Cloning And Expression Of A Putative Exported Protein Coding Gene Sas1089 From An Invasive Community Associated Staphylococcus Aureus Strain Mssa476, Nailah Alzaki

All-Inclusive List of Electronic Theses and Dissertations

Originally, methicillin resistant Staphylococcus aureus (MRSA) occurred primarily in the hospital setting and was designated hospital- associated MRSA (HA-MRSA). In recent years, isolates of Staphylococcus aureus from the community (CA-MRSA), found to be methicillin resistant, are not as resistant tooth~ antibiotics as HA-MRSA. However, community strains are noted for their invasiveness causing soft tissue infections with propensity for invasion to deeper sites.

An Iterative Strategy Combining Biophysical Criteria And Duration Hidden Markov) Models For Structural Predictions Of Chlamydia Trachomatis S66 Promoters, Ronna R. Mallios, David M. Ojcius, David H. Ardell

All Dugoni School of Dentistry Faculty Articles

Background: Promoter identification is a first step in the quest to explain gene regulation in bacteria. It has been demonstrated that the initiation of bacterial transcription depends upon the stability and topology of DNA in the promoter region as well as the binding affinity between the RNA polymerase σ-factor and promoter. However, promoter prediction algorithms to date have not explicitly used an ensemble of these factors as predictors. In addition, most promoter models have been trained on data from Escherichia coli. Although it has been shown that transcriptional mechanisms are similar among various bacteria, it is quite possible that the …

Single-Amino-Acid Alterations In A Highly Conserved Central Region Of Vesicular Stomatitis Virus N Protein Differentially Affect The Viral Nucleocapsid Template Functions, Debasis Nayak, Debasis Panda, Subash C. Das, Ming Lou, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

The nucleocapsid protein (N) of vesicular stomatitis virus and other rhabdoviruses plays a central role in the assembly and template functions of the viral N-RNA complex. The crystal structure of the viral N-RNA complex suggests that the central region of the N protein interacts with the viral RNA. Sequence alignment of rhabdovirus N proteins revealed several highly conserved regions, one of which spanned residues 282 to 291 (GLSSKSPYSS) in the central region of the molecule. Alanine-scanning mutagenesis of this region suggested that replacement of the tyrosine residue at position 289 (Y289) with alanine resulted in an N-RNA template that is …

Effects Of Estrogen On Muscle Damage In Response To An Acute Resistance Exercise Protocol, Megan R. Wolf

Honors Scholar Theses

Creatine Kinase (CK) is used as a measure of exercise-induced muscle membrane damage. During acute eccentric (muscle lengthening) exercise, muscle sarcolemma, sarcoplasmic reticulum, and Z-lines are damaged, thus causing muscle proteins and enzymes to leak into the interstitial fluid.

Strenuous eccentric exercise produces an elevation of oxygen free radicals, which further increases muscle damage. Muscle soreness and fatigue can be attributed to this membrane damage. Estradiol, however, may preserve membrane stability post-exercise (Brancaccio, Maffulli, & Limongelli, 2007; Carter, Dobridge, & Hackney, 2001; Tiidus, 2001). Because estradiol has a similar structure to Vitamin E, which is known to have antioxidant properties, …

Genetic Variations In A Well Conserved 5-Untranslated Region Of Hepatitis C Virus Genome Isolated In Pakistan, Anila Yasmeen, Anwar Ali Siddiqui, Saeed Hamidb, Taranum Sultana, Wasim Jafri, Mats A.A. Perssonc

Department of Biological & Biomedical Sciences

The diversity and extent of sequence variations between hepatitis C virus (HCV) isolates from Pakistan were studied and the probable effects of these variations were assessed on secondary viral structures. Sequencing and phylogenetic analysis was performed on 33 samples, of which 25 were typed as genotype 3 by RFLP (restriction fragment length polymorphism) and 8 remained unresolved. Rooted neighbour-joining (NJ) tree revealed that 28 isolates were HCV type 3a and 5 isolates were typed as 3b. The majority of unresolved samples clustered in a different branch of genotype 3, supported by a bootstrap value of 71%. Another, cluster, cluster I, …