Cell and Developmental Biology Commons^™

Identification Of A Novel Protein Interaction That Elucidates The Mechanism Of Idiopathic Recurrent Miscarriages In Women With Nlrp2 Mutations, Nayeon Son

Graduate Theses/Dissertations

The protein NOD-Like receptor pyrin domain containing 2 (NLRP2) is one member of a larger family of protein receptors that plays an important role in our innate immune system. In humans, the NLR family consists of 22 proteins. However, only about a half of NLRs’ functions are known, but many are pro-inflammatory, causing inflammation. NLRP2 has been identified to be a maternal effect gene regulating early embryo development in idiopathic recurrent miscarriages. In previous studies, mutations in the NLRP2 gene resulted in genetic maternal imprinting disorders due to NLRP2 regulating DNA methylation. However, the exact mechanisms involved in recurrent miscarriages …

Protein Charge Parameters That Influence Stability And Cellular Internalization Of Polyelectrolyte Complex Micelles, Rachel A. Kapelner, Rachel S. Fisher, Shana Elbaum-Garfinkle, Allie C. Obermeyer

Advanced Science Research Center

Proteins are an important class of biologics, but there are several recurring challenges to address when designing protein-based therapeutics. These challenges include: the propensity of proteins to aggregate during formulation, relatively low loading in traditional hydrophobic delivery vehicles, and inefficient cellular uptake. This last criterion is particularly challenging for anionic proteins as they cannot cross the anionic plasma membrane. Here we investigated the complex coacervation of anionic proteins with a block copolymer of opposite charge to form polyelectrolyte complex (PEC) micelles for use as a protein delivery vehicle. Using genetically modified variants of the model protein green fluorescent protein (GFP), …

Identifying The Human Homologs Of Yeast Rab Proteins Ypt10 & Ypt11 And A Global-Scale Louse Endosymbiont Genome Variation, Nathaniel P. Smith

Theses and Dissertations

Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease that causes loss of upper and/or lower motor neurons, and currently has no treatment or cure available. Over 90% of cases occur spontaneously with unknown causes, highlighting the complexity of the disease, and only 10% of cases are linked to heritable genetic mutations. Numerous ALS-linked genes are conserved through evolution, and model organisms may therefore provide opportunities to understand disease pathology at a molecular or cellular level, proving instrumental in identifying therapeutic targets. ALS subtype 8 (ALS8) is caused by an autosomal dominant P56S mutation in the VAPB gene that …

Genetic Underpinnings Of Novel Trait Development In A Euwallacea-Fusarium Mutualism, Elaina J. Spahr

Graduate Theses, Dissertations, and Problem Reports (ETD)

Evolutionary Developmental Biology seeks to answer fundamental questions about the mechanisms underlying the evolution and innovation towards increasing structural complexity within the body plan. The ambrosia symbiosis, a polyphyletic group of xylem-feeding beetles, provides a wealth of diversity in novel pouch-like structures called mycangia. This diversity could serve as a rich model for understanding how mutualisms may prompt structural innovation and diversification in a host organism. The ambrosia symbiosis has become a fast-growing research subject in entomology and forest pathology but has yet to experience the same attention under the lens of evolutionary developmental genetics.

Development of mycangia was examined …

Characterization Of Two Krab-Containing Zinc Finger Transcription Factors In Bovine Preimplantation Embryonic Development, Mingxiang Zhang

Graduate Theses, Dissertations, and Problem Reports (ETD)

Oocyte developmental competence or oocyte intrinsic quality describes the capability of oocytes to resume meiosis, cleave and develop to blastocyst stage after fertilization, implant and develop to term in a good health. A growing number of evidences indicate that the majority of embryonic mortality occurs during early embryonic development in different species, including human, horse and cattle primarily due to poor oocyte quality. Maternal effect genes are key aspects of oocyte quality, which are transcribed during the process of oogenesis and folliculogenesis. The maternal factors are accumulated in oocytes, orchestrating various early developmental events including fertilization, epigenetic reprogramming and zygotic …

Getting To The Root Cause: The Genetic Underpinnings Of Root System Architecture And Rhizodeposition In Sorghum, Farren Smith

Graduate Theses, Dissertations, and Problem Reports (ETD)

Plants are some of the most diverse organisms on earth, consisting of more than 350,000 different species. To understand the underlying processes that contributed to plant diversification, it is fundamental to identify the genetic and genomic components that facilitated various adaptations over evolutionary history. Most studies to date have focused on the underlying controls of above-ground traits such as grain and vegetation; however, little is known about the “hidden half” of plants. Root systems comprise half of the total plant structure and provide vital functions such as anchorage, resource acquisition, and storage of energy reserves. The execution of these key …

Induction Of Il19 Expression Through Jnk And Cgas-Sting Modulates Dna Damage–Induced Cytokine Production, Sara H. Small, E. Jessica Tang, Ryan L. Ragland, Yaroslava Ruzankina, David W. Schoppy, Rahul S. Mandal, M. Rebecca Glineburg, Zgjim Ustelenca, Daniel J. Powell, Fiona Simpkins, F. Bradley Johnson, Eric J. Brown

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Cytokine production is a critical component of cell-extrinsic responses to DNA damage and cellular senescence. Here, we demonstrated that expression of the gene encoding interleukin-19 (IL-19) was enhanced by DNA damage through pathways mediated by c-Jun amino-terminal kinase (JNK) and cGAS-STING and that IL19 expression was required for the subsequent production of the cytokines IL-1, IL-6, and IL-8. IL19 expression was stimulated by diverse cellular stresses, including inhibition of the DNA replication checkpoint kinase ATR (ataxia telangiectasia and Rad3-related protein), oncogene expression, replicative exhaustion, oxidative stress, and DNA double-strand breaks. Unlike the production of IL-6 and IL-8, IL19 expression was …

Functional Characterization Of Cancer-Associated Dna Polymerase Ε Variants, Stephanie R. Barbari

Theses & Dissertations

Replicative DNA polymerases ε (Polε) and δ (Polδ) achieve high fidelity DNA synthesis through a precise balance of polymerization and exonucleolytic proofreading. Errors that escape proofreading are corrected by DNA mismatch repair (MMR). Ultramutated human cancers with proficient MMR carry alterations in the exonuclease domain of Polε, which were initially predicted to abolish proofreading. However, functional studies in yeast of the most recurrent Polε-P286R variant suggested defects beyond a loss of exonuclease activity. Indeed, biochemical analysis of the yeast Polε-P286R analog revealed increased polymerization capacity in addition to decreased proofreading, which enables efficient mismatch extension and bypass of replication-blocking non-B …

Citric Acid/Β-Alanine Carbon Dots As A Novel Tool For Delivery Of Plasmid Dna Into E. Coli Cells, Anju Pandey, Asmita Devkota, Anil Sigdel, Zeinab Yadegari, C. Korsi Dumenyo, Ali Taheri

Agricultural and Environmental Sciences Faculty Research

Successful delivery of plasmid DNA into the microbial cells is fundamental in recombinant DNA technology. Natural bacterial transformation is limited to only certain species due in part to the repulsive forces between negatively charged DNA and bacterial membranes. Most common method of DNA delivery into bacteria is artificial transformation through heat shock and electroporation. These methods require sophisticated instruments and tedious steps in preparation of competent cells. Transformation by conjugation is also not applicable to all plasmids. Nanoparticles have been used successfully in therapeutics for drug delivery into animal cells. They are starting to gain popularity in plant sciences as …

Co-Option Of The Yolkless Oocyte Receptor For Crispr/Cas9 May Induce Transient Sterility In Female Drosophila Grimshawi Flies, Bronwyn Miller

LSU New Orleans Theses and Dissertations

Evolutionary developmental biology has historically used generalist model organisms like Drosophila melanogaster. The Hawaiian Drosophilidae radiation, which occurred just 25 million years ago, is an ideal lineage for work in Evo-Devo. Hawaiian flies make up 25% of the world’s Drosophila species and extreme diversity is seen throughout the group. D. grimshawi is a generalist Hawaiian picture-wing fly that has served as a model for the Hawaiian Drosophilidae radiation. However, D. grimshawi’s power and use as a model organism is constrained by our lack of ability to manipulate its genome. In this work, I attempted to edit the genome of …

Mutant Kras Alters Extracellular Vesicle Microrna Sorting In Pancreatic Cystic Neoplasms, Rachel L. Dittmar

Dissertations and Theses (Open Access)

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers by organ site with a 5-year survival rate of just 10.8%. This is largely because most patients do not experience symptoms until the disease has already metastasized. The best hope to cure PDAC is surgery, which can only be done with a curative intent at an early stage when the disease is localized. There are no reliable circulating, body-fluid-based biomarkers to detect early stage PDAC or its precursor lesions in a timely manner for effective surgical intervention. When potential PDAC precursor lesions, such as mucinous pancreatic cysts are found, there are …

Bicuspid Aortic Valve In Pregnancy Complicated By Aortic Valve Vegetation, Aortic Root Abscess, And Aortic Insufficiency, Shan He, Christopher A Huynh, Yi Deng, Sandeep Markan, Anvinh Nguyen

Faculty, Staff and Students Publications

A 39-year-old patient presented to our Labor and Delivery unit with fever and nausea in the context of recent bacteriuria. She was found to be in sepsis due to an incidental bicuspid aortic valve (BAV) complicated by aortic valvular vegetations, severe aortic insufficiency, and aortic root abscess, requiring an emergent cesarean section. Three days after delivery, the patient successfully underwent a mechanical aortic valve replacement and root reconstruction. In this case report, medical, surgical, and anesthetic management of parturient patients with BAV are discussed. The management of this congenital valvulopathy and vasculopathy is complicated by the extensive hemodynamic and cardiovascular …

The Multifaceted Role Of Ccar-1 In The Alternative Splicing And Germline Regulation In Caenorhabditis Elegans, Doreen Ikhuva Lugano

USF Tampa Graduate Theses and Dissertations

The Cell Division Cycle and Apoptosis Regulator (CCAR) family members are an enigmatic family of proteins regulating metabolism, cancer, apoptosis, DNA damage, and stress. Mammals have CCAR family members, CCAR1 and CCAR2/DBC1, which evolved from the founding family member CCAR-1/LST-3 expressed in Caenorhabditis elegans. Several studies have shown the importance of understanding these proteins' function in standard and altered physiological processes. Our studies aim to understand the genome-wide alternative splicing and germline regulation of Caenorhabditis elegans CCAR-1 in normal and heat shock conditions. Recently, mammalian CCAR family member CCAR2/DBC1 regulates the alternative splicing by forming a complex with ZNF326. This …

Regulatory Non-Coding Rnas Modulate Transcriptional Activation During B Cell Development, Mary Attaway, Tzippora Chwat-Edelstein, Bao Q. Vuong

Publications and Research

B cells play a significant role in the adaptive immune response by secreting immunoglobulins that can recognize and neutralize foreign antigens. They develop from hematopoietic stem cells, which also give rise to other types of blood cells, such as monocytes, neutrophils, and T cells, wherein specific transcriptional programs define the commitment and subsequent development of these different cell lineages. A number of transcription factors, such as PU.1, E2A, Pax5, and FOXO1, drive B cell development. Mounting evidence demonstrates that non-coding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), modulate the expression of these transcription factors directly by binding …

Human 5’-Tailed Mirtrons Are Processed By Rnasep, Mohammad Farid Zia

Dissertations

Approximately a thousand microRNAs (miRNAs) are documented from human cells. A third appear to transit non-canonical pathways that typically bypass processing by Drosha, the dedicated nuclear miRNA producing enzyme. The largest class of non-canonical miRNAs are mirtrons which eschew Drosha to mature through spliceosome activity. While mirtrons are found in several configurations, the vast majority of human mirtron species are 5’-tailed. For these mirtrons, a 3’ splice site defines the 3’ end of their hairpin precursor while a “tail” of variable length separates the 5’ base of the hairpin from the nearest splice site. How this tail is removed is …

Determining The Primary Dna Substrates Of Shld2'S Ob-Fold Domains, Hari Patchigolla

Holster Scholar Projects

Failure to repair DNA double-stranded breaks leads to cell death. Radiation therapy is commonly used to kill cancer cells by inducing these breaks. However resistance to radiation therapy, due to a hyperactive DNA double-stranded break repair pathway, is a common occurrence that makes cancer patients more prone to relapse. The Shieldin complex is shown to promote DNA-double stranded break repair by binding to DNA at sites of damage. Thus, the objective of this project is to understand the affinity and type of DNA that Shieldin binds to, through gel-shift assays, for the eventual creation of an inhibitor for this protein …

Neuron-Specific Mitochondrial Oxidative Stress Results In Epilepsy, Glucose Dysregulation And A Striking Astrocyte Response, Ruth E Fulton, Jennifer N Pearson-Smith, Christopher Q Huynh, Timothy Fabisiak, Li-Ping Liang, Stefanos Aivazidis, Brigit A High, Georgia Buscaglia, Timothy Corrigan, Robert Valdez, Takahiko Shimizu, Manisha N Patel

Faculty, Staff and Students Publications

Mitochondrial superoxide (O2.−) production is implicated in aging, neurodegenerative disease, and most recently epilepsy. Yet the specific contribution of neuronal O2.− to these phenomena is unclear. Here, we selectively deleted superoxide dismutase-2 (SOD2) in neuronal basic helix-loop-helix transcription factor (NEX)-expressing cells restricting deletion to a subset of excitatory principle neurons primarily in the forebrain (cortex and hippocampus). This resulted in nSOD2 KO mice that lived into adulthood (2-3 months) with epilepsy, selective loss of neurons, metabolic rewiring and a marked mitohormetic gene response. Surprisingly, expression of an astrocytic gene, glial fibrillary acidic protein (GFAP) was significantly increased relative …

Using Molecular Diagnostics To Develop Therapeutic Strategies For Carbapenem-Resistant Gram-Negative Infections, Fred C. Tenover

Biology Faculty Publications

Infections caused by multidrug-resistant Gram-negative organisms have become a global threat. Such infections can be very difficult to treat, especially when they are caused by carbapenemase-producing organisms (CPO). Since infections caused by CPO tend to have worse outcomes than non-CPO infections, it is important to identify the type of carbapenemase present in the isolate or at least the Ambler Class (i.e., A, B, or D), to optimize therapy. Many of the newer beta-lactam/beta-lactamase inhibitor combinations are not active against organisms carrying Class B metallo-enzymes, so differentiating organisms with Class A or D carbapenemases from those with Class B enzymes rapidly …

The Effects Of Ppal-1 In Arabidopsis Gamete Development, Amanda J White, Susana Perez-Martinez, Mark P. Running

The Cardinal Edge

Prenylation is a type of post-translational modification in which a 15- or 20-carbon lipid is added to the carboxyl (C) terminus of the protein. Arabidopsis thaliana contains the PROTEIN PRENYLTRANSFERASE ALPHA SUBUNIT-LIKE (PPAL) gene, which encodes a protein with homology to the α-subunits of the three known prenylation enzymes, PFT, PGGT, and Rab-GGT. We previously identified two mutations in PPAL, one of which is ppal-1, which contains a T-DNA insertion in the fourth intron. We have previously observed that self-fertilizing heterozygous ppal-1 plants produce progeny in which homozygous ppal-1 is underrepresented. This project attempts to ascertain …

Phenotypic/Genotypic Profile Of Oxa-10-Like-Harboring, Carbapenem-Resistant Pseudomonas Aeruginosa: Using Validated Pharmacokinetic/Pharmacodynamic In Vivo Models To Further Evaluate Enzyme Functionality And Clinical Implications, Fred C. Tenover, Christian M. Gill, Adrian Brink, Chun Yat Chu, Jennifer Coetzee, George Dimopoulos, Clinton Moodley, Christoffel Johannes Opperman, Spyros Pournaras, Isabella A. Tickler, Hafsah Deepa Tootla, Sophia Vourli, David P. Nicolau

Biology Faculty Publications

In vitro MICs and in vivo pharmacodynamics of ceftazidime and cefepime human-simulated regimens (HSR) against modified carbapenem inactivation method (mCIM)-positive Pseudomonas aeruginosa isolates harboring different OXA-10-like subtypes were described. The murine thigh model assessed ceftazidime (2 g every 8 h [q8h] HSR) and cefepime (2 g and 1 g q8h HSR). Phenotypes were similar despite possessing OXA-10-like subtypes with differing spectra. Ceftazidime produced ≥1-log10 killing in all isolates. Cefepime activity was dose dependent and MIC driven. This approach may be useful in assessing the implications of β-lactamase variants.

Developmental Programming Of Dna Methylation And Gene Expression Patterns Is Associated With Extreme Cardiovascular Tolerance To Anoxia In The Common Snapping Turtle, Ilan Ruhr, Jacob Bierstedt, Turk Rhen, Debojyoti Das, Sunil Kumar Singh, Soleille Miller, Dane A. Crossley Ii, Gina L. J. Galli

Biology Faculty Publications

Background

Environmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia …

Investigation Of Notch Signaling In Cone Fate Specification In Vertebrate Retina, Xueqing Chen

Dissertations, Theses, and Capstone Projects

In the vertebrate retina, cone photoreceptors are crucial for high acuity color vision. Several retinal diseases lead to loss of cones and there is a need to identify the normal developmental genesis of these cells to inform the development of stem cell-based therapies. Cone genesis has previously been shown to be repressed by Notch signaling, however, the mechanism by which Notch signaling controls cone fate determination is still unclear. It has been identified that cone photoreceptors are formed from multipotent retinal progenitor cells (RPCs) that first generate genetically-defined, restricted RPCs with limited mitotic and fate potential to preferentially form cones …

The Slit-Binding Ig1 Domain Is Required For Multiple Axon Guidance Activities Of Drosophila Robo2, Lafreda J. Howard, Marie C. Reichert, Timonthy A. Evans

Biological Sciences Faculty Publications and Presentations

Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. Robo receptors signal midline repulsion in response to Slit ligands, which bind to the N-terminal Ig1 domain in most family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 signal Slit-dependent midline repulsion, while Robo2 also regulates the medial-lateral position of longitudinal axon pathways and acts non-autonomously to promote midline crossing of commissural axons. While Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2's other activities are also Slit-dependent. To determine which of Robo2's axon guidance roles …

Vitamin D Modulates Rett Syndrome Phenotypes And Underlying Cellular Pathways In An Mecp2-Mutant Mouse Model, Mayara De Cassia Ribeiro

Dissertations - ALL

Rett syndrome (RTT) is a progressive and severe X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MECP2. There is currently no effective treatment for RTT. Female RTT patients develop relatively normally during the first 6-18 months of life, after which they undergo a period of rapid regression, losing the ability to talk, walk and purposefully use their hands, in addition to suffering from deceleration of head growth, and onset of repetitive, autistic behaviors. RTT symptoms can be partially reversed by the re-expression of Mecp2 in adult mice, suggesting the potential for post-symptomatic therapeutic intervention. Among the many dysfunctions …

Gene Expression Profiling Of Mapk Pathway Inhibitor Resistance In Cutaneous Melanoma: Can Bioinformatics Be Used To Select Better Melanoma Cell Lines?, Stephen Luebker

Theses & Dissertations

Melanoma is the deadliest form of skin cancer, and incidence has continued to increase. Half of all melanomas have a BRAF V600E mutation and respond to MAPK pathway inhibitors, including BRAF inhibitor therapy or BRAF/MEK inhibitor combination therapy, but nearly all patients develop treatment resistance. Melanoma cell lines produce variable results as models of MAPK pathway inhibitor resistance. To better understand how the genomic similarity of a melanoma cell line to patient-derived tumors affects resistance mechanisms, differences in DNA mutations and copy-number alterations were compared between melanoma cell lines profiled by the Cancer Cell Line Encyclopedia and cutaneous melanoma tumors …

Unbiased Automated Quantitation Of Ros Signals In Live Retinal Neurons Of Drosophila Using Fiji/Imagej, Prajakta Deshpande, Neha Gogia, Anuradha Venkatakrishnan Chimata, Amit Singh

Biology Faculty Publications

Numerous imaging modules are utilized to study changes that occur during cellular processes. Besides qualitative (immunohistochemical) or semiquantitative (Western blot) approaches, direct quantitation method(s) for detecting and analyzing signal intensities for disease(s) biomarkers are lacking. Thus, there is a need to develop method(s) to quantitate specific signals and eliminate noise during live tissue imaging. An increase in reactive oxygen species (ROS) such as superoxide (O2•-) radicals results in oxidative damage of biomolecules, which leads to oxidative stress. This can be detected by dihydroethidium staining in live tissue(s), which does not rely on fixation and helps prevent stress on tissues. However, …

Effects Of Trans-Acting Factors On The Translational Machinery In Yeast, Brandon M. Trainor

Graduate School of Biomedical Sciences Theses and Dissertations

Synthesis of proteins, or translation, is a complex biological process requiring the coordinated effort of numerous protein and RNA factors. Central to translation is the ribosome, a complex macromolecular complex consisting of both ribosomal RNA (rRNA) and ribosomal protein (r-protein). Ribosomes are essential and are one of the oldest and most abundant biomolecules across all forms of life. In addition to the ribosome, translation requires messenger RNA (mRNA), transfer-RNA conjugated to an amino acid (aa-tRNA), translation factors, and energy in the form of ATP and GTP. Translation universally occurs in four major stages, initiation, elongation, termination, and recycling, with initiation …

Epithelial Memory Of Resolved Inflammation Limits Tissue Damage While Promoting Pancreatic Tumorigenesis, I-Lin Ho

Dissertations and Theses (Open Access)

Inflammation is a major risk factor for pancreatic ductal adenocarcinoma. When occurring in the context of pancreatitis, mutations of KRAS accelerate tumor development. We discovered that long after its complete resolution, a transient inflammatory event primes pancreatic epithelial cells to subsequent transformation by oncogenic KRAS. Upon recovery from acute inflammation, epithelial cells of the pancreas display an enduring adaptive response associated with sustained transcriptional and epigenetic reprogramming. Such adaptation enables the prompt reactivation of acinar-to-ductal metaplasia (ADM) upon subsequent inflammatory events, thus efficiently limiting tissue damage via rapid decrease of zymogen production. We propose that since activating mutations of KRAS …

Npsd4: A New Player In Sumo-Dependent Dna Repair, Erin Atkinson

Dissertations and Theses (Open Access)

The human genome is under constant threat from sources of damage and stress. Improper resolution of DNA damage lesions can lead to mutations, oncogene activation, and genomic instability. Difficult-to-replicate-loci present barriers to DNA replication that, when not properly resolved, lead to replication fork stalling and collapse and genomic instability.

DNA damage and replication stress trigger signaling cascades potentiated by multiple types of post-translational modifications, including SUMOylation. Through proteomic analysis of proteins involved in SUMOylation following DNA damage, our lab identified an uncharacterized protein that we named New Player in SUMO-dependent DNA damage repair 4 (NPSD4). Through an additional proteomic screen, …

Targeting Plasma Membrane Phosphatidylserine Content To Inhibit Oncogenic Kras Function, Walaa E. Kattan

Dissertations and Theses (Open Access)

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We therefore investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidylinositol-4-phosphate (PI4P) synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion …