Phase separation of YAP-MAML2 differentially regulates the transcriptome.

Phase separation (PS) drives the formation of biomolecular condensates that are emerging biological structures involved in diverse cellular processes. Recent studies have unveiled PS-induced formation of several transcriptional factor (TF) condensates that are transcriptionally active, but how strongly PS promotes gene activation remains unclear. Here, we show that the oncogenic TF fusion Yes-associated protein 1-Mastermind like transcriptional coactivator 2 (YAP-MAML2) undergoes PS and forms liquid-like condensates that bear the hallmarks of transcriptional activity. Furthermore, we examined the contribution of PS to YAP-MAML2-mediated gene expression by developing a chemogenetic tool that dissolves TF condensates, allowing us to compare phase-separated and non-phase-separated conditions at identical YAP-MAML2 protein levels. We found that a small fraction of YAP-MAML2-regulated genes is further affected by PS, which include the canonical YAP target genes CTGF and CYR61, and other oncogenes. On the other hand, majority of YAP-MAML2-regulated genes are not affected by PS, highlighting that transcription can be activated effectively by diffuse complexes of TFs with the transcriptional machinery. Our work opens new directions in understanding the role of PS in selective modulation of gene expression, suggesting differential roles of PS in biological processes.

Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter.

Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.

Targeting SWI/SNF ATPases in H3.3K27M diffuse intrinsic pontine gliomas.

Diffuse midline gliomas (DMGs) including diffuse intrinsic pontine gliomas (DIPGs) bearing lysine-to-methionine mutations in histone H3 at lysine 27 (H3K27M) are lethal childhood brain cancers. These tumors harbor a global reduction in the transcriptional repressive mark H3K27me3 accompanied by an increase in the transcriptional activation mark H3K27ac. We postulated that H3K27M mutations, in addition to altering H3K27 modifications, reprogram the master chromatin remodeling switch/sucrose nonfermentable (SWI/SNF) complex. The SWI/SNF complex can exist in two main forms termed BAF and PBAF that play central roles in neurodevelopment and cancer. Moreover, BAF antagonizes PRC2, the main enzyme catalyzing H3K27me3. We demonstrate that H3K27M gliomas show increased protein levels of the SWI/SNF complex ATPase subunits SMARCA4 and SMARCA2, and the PBAF component PBRM1. Additionally, knockdown of mutant H3K27M lowered SMARCA4 protein levels. The proteolysis targeting chimera (PROTAC) AU-15330 that simultaneously targets SMARCA4, SMARCA2, and PBRM1 for degradation exhibits cytotoxicity in H3.3K27M but not H3 wild-type cells. AU-15330 lowered chromatin accessibility measured by ATAC-Seq at nonpromoter regions and reduced global H3K27ac levels. Integrated analysis of gene expression, proteomics, and chromatin accessibility in AU-15330-treated cells demonstrated reduction in the levels of FOXO1, a key member of the forkhead family of transcription factors. Moreover, genetic or pharmacologic targeting of FOXO1 resulted in cell death in H3K27M cells. Overall, our results suggest that H3K27M up-regulates SMARCA4 levels and combined targeting of SWI/SNF ATPases in H3.3K27M can serve as a potent therapeutic strategy for these deadly childhood brain tumors.

Ab initio study of changing the oxygen reduction activity of Co-Fe-based perovskites by tuning the B-site composition.

Perovskite oxides are promising low-cost and stable alternative electrocatalysts for the oxygen reduction reaction (ORR), relative to the precious metal-based electrocatalysts. Despite the experimental research on substituting various transition metals into the B-site of perovskite catalysts to improve the ORR performance, the detailed ORR mechanism due to the substitution process is rarely studied. In this paper, the ORR activity of La0.5Sr0.5CoxFe1-xO3 perovskites (x = 0, 0.25, 0.5, 0.75, and 1) is studied by density functional theory (DFT). The ORR mechanism in alkaline solution is theoretically examined as a function of the Co/Fe composition at different potentials. The substitution of Co for Fe at the B-site of the perovskites dramatically changes the theoretical overpotential and enhances the activity. The HOO* formation is the potential-determining step for all the Co/Fe compositions. In comparison with the other compositions, the Co0.5/Fe0.5 composition exhibits the lowest overpotential and bonding with the reaction intermediates moderately. Furthermore, the oxygen binding energy is correlated with the bulk oxygen p-band center relative to the Fermi level. Among all the Co/Fe compositions, the Co0.5/Fe0.5 composition shows neither too low nor too high oxygen p-band center value. These results provide deep insights into the ORR mechanism on B-site substituted perovskites and guidelines for the design of cost-effective and Pt-free electrocatalysts for oxygen reduction.

Impedimetric sensing platform for sensitive carbendazim detection using MOCVD-synthesized copper graphene.

Nanostructures of graphene were synthesized for electrochemical carbendazim (CBZ) fungicide detection via metal-organic chemical vapor deposition (MOCVD). The arduous process of graphene transfer is eliminated by this innovative approach to MOCVD graphene development. It also generates several defects and impurities and ultimately leads to the uniform deposition of graphene on SiO2/Si. SEM, EDX, and ICP-AES were used to assess the morphological properties and chemical composition of the materials. To obtain in-depth knowledge of the entire system, the electrochemical behavior was also investigated using voltammetric techniques and electrochemical impedance spectroscopy. The interaction of particles of copper with CBZ and the enhanced surface area of graphene, which causes a strong oxidation current, has been demonstrated to achieve the ideal CBZ sensing behavior. The electrode responded linearly at CBZ concentration levels of 1 to 50 nM, and the sensitivity of the sensing materials was estimated to be 0.0337 Ω nM-1. The statistical analysis validates the electrode's exceptional selectivity and remarkable reproducibility in determining CBZ.

Comparison of Postoperative Nausea and Vomiting Incidence between Remimazolam and Sevoflurane in Tympanoplasty with Mastoidectomy: A Single-Center, Double-Blind, Randomized Controlled Trial.

Background and Objectives: Postoperative nausea and vomiting (PONV) is a common adverse effect of general anesthesia, especially in middle ear surgery. Remimazolam is a newer benzodiazepine recently approved for use in general anesthesia. This study aimed to compare the incidence rate of PONV after tympanoplasty with mastoidectomy between using remimazolam and sevoflurane. Materials and Methods: This study included 80 patients undergoing elective tympanoplasty with mastoidectomy. The patients were randomly assigned to either the remimazolam or sevoflurane group. The primary outcome was the incidence rate of PONV 12 h after surgery. The secondary outcomes were the incidence rate of PONV 12-24 and 24-48 h after surgery, severity of PONV, incidence rate of vomiting, administration of rescue antiemetics, hemodynamic stability, and recovery profiles. Results: The incidence rate of PONV 0-12 h after tympanoplasty with mastoidectomy was significantly lower in the remimazolam group compared with that in the sevoflurane group (28.9 vs. 57.9%; p = 0.011). However, the incidence rate of delayed PONV did not differ between the two groups. PONV severity in the early periods after the surgery was significantly lower in the remimazolam group than in the sevoflurane group. The incidence rate of adverse hemodynamic events was lower in the remimazolam group than in the sevoflurane group, but there was no difference in the overall trends of hemodynamic data between the two groups. There was no difference in recovery profiles between the two groups. Conclusions: Remimazolam can significantly reduce the incidence rate of early PONV after tympanoplasty with mastoidectomy under general anesthesia.

Interfacial Strain-Modulated Nanospherical Ni2 P by Heteronuclei-Mediated Growth on Ti3 C2 Tx MXene for Efficient Hydrogen Evolution.

Interface modulation of nickel phosphide (Ni2 P) to produce an optimal catalytic activation barrier has been considered a promising approach to enhance the hydrogen production activity via water splitting. Herein, heteronuclei-mediated in situ growth of hollow Ni2 P nanospheres on a surface defect-engineered titanium carbide (Ti3 C2 Tx ) MXene showing high electrochemical activity for the hydrogen evolution reaction (HER) is demonstrated. The heteronucleation drives intrinsic strain in hexagonal Ni2 P with an observable distortion at the Ni2 P@Ti3 C2 Tx MXene heterointerface, which leads to charge redistribution and improved charge transfer at the interface between the two components. The strain at the Ni2 P@Ti3 C2 Tx MXene heterointerface significantly boosts the electrochemical catalytic activities and stability toward HER in an acidic medium via a combination between experimental results and theoretical calculations. In a 0.5 m H2 SO4 electrolyte, the Ni2 P@Ti3 C2 Tx MXene hybrid shows excellent HER catalytic performance, requiring an overpotential of 123.6 mV to achieve 10 mA cm-2 with a Tafel slope of 39 mV dec-1 and impressive durability over 24 h operation. This approach presents a significant potential to rationally design advanced catalysts coupled with 2D materials and transition metal-based compounds for state-of-the-art high efficiency energy conversions.

Comparison among three different intensities of eccentric contractions of the elbow flexors resulting in the same strength loss at one day post-exercise for changes in indirect muscle damage markers.

PURPOSE: The present study compared three different intensity elbow flexor eccentric exercises resulting in the same magnitude of maximal voluntary isomeric contraction torque (MVC) decrease at 1 day post-exercise for changes in several indirect markers of muscle damage and proprioception. METHODS: Sedentary young men performed eccentric contractions of the elbow flexors using a dumbbell corresponding to either 10%, 50% or 100% of MVC to induce ~ 20% decrease in MVC at 1 day post-exercise (n = 12/group). MVC, range of motion (ROM), upper arm circumference (CIR), muscle soreness, plasma creatine kinase (CK) activity, and proprioception measures (force match, joint position sense) were taken before to 5 days after exercise, and the changes were compared among the groups. RESULTS: MVC and ROM recovered faster (P < 0.05) in the order of 10% (e.g., 3 days post-exercise: - 3 ± 4%, 0 ± 1%), 50% (- 12 ± 3%, - 3 ± 2%) and 100% group (- 16 ± 4%, - 5 ± 1%). Peak CIR, muscle soreness and CK activity were smaller for 10% (Δ3 ± 1 mm, 26 ± 10 mm, 1049 ± 316 IU/L) than 50% (Δ6 ± 2 mm, 36 ± 14 mm, 1473 ± 429 IU/L) and 100% groups (Δ8 ± 2 mm, 47 ± 15 mm, 2104 ± 929 IU/L). The proprioception measures recovered faster (P < 0.05) for 10% followed by 50% then 100% group. The recovery rate of MVC from immediately to 1 day post-exercise was correlated (P < 0.05) with the changes in the muscle damage and proprioception markers. CONCLUSION: These results suggest that the MVC at 1 day post-exercise does not necessarily predict the changes in muscle damage markers in the following days, but the MVC recovery rate in the first 24 h reflects the magnitude of muscle damage better.

Psychosocial Support during the COVID-19 Outbreak in Korea: Activities of Multidisciplinary Mental Health Professionals.

As of April 18, 2020, there have been a total of 10,653 confirmed cases and 232 deaths due to coronavirus disease 2019 (COVID-19) in Korea. The pathogen spread quickly, and the outbreak caused nationwide anxiety and shock. This study presented the anecdotal records that provided a detailed process of the multidisciplinary teamwork in mental health during the COVID-19 outbreak in the country. Psychosocial support is no less important than infection control during an epidemic, and collaboration and networking are at the core of disaster management. Thus, a multidisciplinary team of mental health professionals was immediately established and has collaborated effectively with its internal and external stakeholders for psychosocial support during the COVID-19 outbreak.

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease.

Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.

Genetic and pharmacological evidence implicates cathepsins in Niemann-Pick C cerebellar degeneration.

Niemann-Pick C1 (NPC) disease, an autosomal recessive lipid trafficking disorder caused by loss-of-function mutations in the NPC1 gene, is characterized by progressive neurodegeneration resulting in cognitive impairment, ataxia and early death. Little is known about the cellular pathways leading to neuron loss. Here, we studied the effects of diminishing expression of cystatin B, an endogenous inhibitor of cathepsins B, H and L, on the development of NPC neuropathology. We show that decreased expression of cystatin B in patient fibroblasts enhances cathepsin activity. Deletion of the encoding Cstb gene in Npc1-deficient mice resulted in striking deleterious effects, particularly within the cerebellum where diffuse loss of Purkinje cells was observed in young mice. This severe pathology occurred through cell autonomous mechanisms that triggered Purkinje cell death. Moreover, our analyses demonstrated the mislocalization of lysosomal cathepsins within the cytosol of Npc1-deficient Purkinje cells. We provide evidence that this may be a consequence of damage to lysosomal membranes by reactive oxygen species (ROS), leading to the leakage of lysosomal contents that culminates in apoptotic cell death. Consistent with this notion, toxicity from ROS was attenuated in an NPC cell model by cystatin B over-expression or pharmacological inhibition of cathepsin B. The observation that Npc1 and Cstb deletion genetically interact to potently enhance the degenerative phenotype of the NPC cerebellum provides strong support for the notion that lysosomal membrane permeabilization contributes to cerebellar degeneration in NPC disease.

Dynamic Imaging of Small Molecule Induced Protein-Protein Interactions in Living Cells with a Fluorophore Phase Transition Based Approach.

Protein-protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule induced protein-protein association and dissociation in living cells. Here we have applied a fluorophore phase transition based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drug (IMiD) induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiD analogue (e.g., CC-885) induced PPI between cereblon and GSPT1. Furthermore, SPPIER can visualize bifunctional molecules (e.g. PROTAC)-induced PPI between an E3 ubiquitin ligase and a target protein. Lastly, SPPIER can be modified to image small molecule induced protein-protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intense brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.

One-step isothermal detection of multiple KRAS mutations by forming SNP specific hairpins on a gold nanoshell.

We developed a one-step isothermal method for typing multiple KRAS mutations using a designed set of primers to form a hairpin on a gold nanoshell upon being ligated by a SNP specific DNA ligase after binding of targets. As a result, we could detect as low as 20 attomoles of KRAS mutations within 1 h.

Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5-SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51-SWI5-SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells.

In search of the vector(s) of Babesia rossi in Nigeria: molecular detection of B. rossi DNA in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) ticks collected from dogs, circumstantial evidence worth exploring.

The brown dog tick Rhipicephalus sanguineus (sensu lato) (Acari: Ixodidae) has a cosmopolitan distribution, is a proven vector of a host of pathogens with emerging evidence incriminating it in the transmission of some others. Specifically it is reputed as the main vector of Babesia vogeli whereas the southern African yellow dog tick Haemaphysalis elliptica, long considered to be H. leachi, is apparently the only proven vector of B. rossi, since the resurrection of the separate species H. elliptica as a member of the leachi-group by Apanaskevich et al. However, recent epidemiological surveys conducted in Nigeria show higher prevalence of B. rossi than B. vogeli infection in dogs most of whom were infested with R. sanguineus and rarely with ticks of the H. leachi group. The discrepancy between tick distribution and Babesia spp. prevalent in dogs stimulated us to investigate the possible role of R. sanguineus (s.l.) in the natural transmission of B. rossi. Out of a total of 66 tick samples identified morphologically and molecularly as R. sanguineus collected from dogs manifesting clinical signs of tick-borne diseases, eight (12%) were positive in nested PCR for Babesia sp. DNA. Sequencing results for these amplified products showed that all of the 18S rDNA sequences (693 bp) were identical to each other, and bore 99.3-99.9% identities with those from other B. rossi isolates accessible in GenBank. None of the ticks harbored the DNA of B. vogeli or B. canis. The possible implications for the detection of B. rossi DNA in R. sanguineus (s.l.) ticks collected from dogs in the epidemiology of B. rossi infection of dogs in Nigeria is highlighted.

Development of a Quenchbody for the Detection and Imaging of the Cancer-Related Tight-Junction-Associated Membrane Protein Claudin.

Claudins (CLs) are membrane proteins found in tight junctions and play a major role in establishing the intercellular barrier. However, some CLs are abnormally overexpressed on tumor cells and are valid clinical biomarkers for cancer diagnosis. Here, we constructed antibody Fab fragment-based Quenchbodies (Q-bodies) as effective and reliable fluorescent sensors for detecting and visualizing CLs on live tumor cells. The variable region genes for anti-CL1 and anti-CL4 antibodies were used to express recombinant Fab fragments, and clones recognizing CL4 with high affinity were selected for making Q-bodies. When two fluorescent dyes were conjugated to the N-terminal tags attached to the Fab, the fluorescent signal was significantly increased after adding nanomolar-levels of purified CL4. Moreover, addition of the Q-body to CL4-expressing cells including CL4-positive cancer cells led to a clear fluorescence signal with low background, even without washing steps. Our findings suggested that such Q-bodies would serve as a potent tool for specifically illuminating membrane targets expressed on cancer cells, both in vitro and in vivo.

The Pine-Needle-Inspired Structure of Zinc Oxide Nanorods Grown on Electrospun Nanofibers for High-Performance Flexible Supercapacitors.

Flexible supercapacitors with high electrochemical performance and stability along with mechanical robustness have gained immense attraction due to the substantial advancements and rampant requirements of storage devices. To meet the exponentially growing demand of microsized energy storage device, a cost-effective and durable supercapacitor is mandatory to realize their practical applications. Here, in this work, the fabrication route of novel electrode materials with high flexibility and charge-storage capability is reported using the hybrid structure of 1D zinc oxide (ZnO) nanorods and conductive polyvinylidene fluoride-tetrafluoroethylene (P(VDF-TrFE)) electrospun nanofibers. The ZnO nanorods are conformably grown on conductive P(VDF-TrFE) nanofibers to fabricate the light-weighted porous electrodes for supercapacitors. The conductive nanofibers acts as a high surface area scaffold with significant electrochemical performance, while the addition of ZnO nanorods further enhances the specific capacitance by 59%. The symmetric cell with the fabricated electrodes presents high areal capacitance of 1.22 mF cm-2 at a current density of 0.1 mA cm-2 with a power density of more than 1600 W kg-1 . Furthermore, these electrodes show outstanding flexibility and high stability with 96% and 78% retention in specific capacitance after 1000 and 5000 cycles, respectively. The notable mechanical durability and robustness of the cell acquire both good flexibility and high performance.

Immunohistochemical analysis of H3K27me3 demonstrates global reduction in group-A childhood posterior fossa ependymoma and is a powerful predictor of outcome.

Posterior fossa ependymomas (EPN_PF) in children comprise two morphologically identical, but biologically distinct tumor entities. Group-A (EPN_PFA) tumors have a poor prognosis and require intensive therapy. In contrast, group-B tumors (EPN_PFB) exhibit excellent prognosis and the current consensus opinion recommends future clinical trials to test the possibility of treatment de-escalation in these patients. Therefore, distinguishing these two tumor subtypes is critical. EPN_PFA and EPN_PFB can be distinguished based on DNA methylation signatures, but these assays are not routinely available. We have previously shown that a subset of poorly prognostic childhood EPN_PF exhibits global reduction in H3K27me3. Therefore, we set out to determine whether a simple immunohistochemical assay for H3K27me3 could be used to segregate EPN_PFA from EPN_PFB tumors. We assembled a cohort of 230 childhood ependymomas and H3K27me3 immunohistochemistry was assessed as positive or negative in a blinded manner. H3K27me3 staining results were compared with DNA methylation-based subgroup information available in 112 samples [EPN_PFA (n = 72) and EPN_PFB tumors (n = 40)]. H3K27me3 staining was globally reduced in EPN_PFA tumors and immunohistochemistry showed 99% sensitivity and 100% specificity in segregating EPN_PFA from EPN_PFB tumors. Moreover, H3K27me3 immunostaining was sufficient to delineate patients with worse prognosis in two independent, non-overlapping cohorts (n = 133 and n = 97). In conclusion, immunohistochemical evaluation of H3K27me3 global reduction is an economic, easily available and readily adaptable method for defining high-risk EPN_PFA from low-risk posterior fossa EPN_PFB tumors to inform prognosis and to enable the design of future clinical trials.

An open sandwich immunoassay for detection of 13(R,S)-hydroxy-9(E),11(E)-octadecadienoic acid.

Lipid peroxidation is involved in many disorders and diseases such as cardiovascular disease, cancers, neurodegenerative diseases, and even aging. Lipid peroxidation products existing in blood or bodily fluids are very important biomarkers for the diagnosis of such diseases. In particular, 13(R,S)-hydroxy-9(E),11(E)-octadecadienoic acid (13-(E,E)-HODE) is an oxidiation product of linoleic acid, which is an important biomarker for many diseases such as diabetes and Alzheimer's disease. In this study, we successfully displayed the antigen-binding fragment of an antibody produced by hybridoma 1213-1 on the M13 phage and performed analysis of the antibody variable region genes. The blast results suggested that it is a novel antibody. We also developed a phage-antibody-based competitive ELISA and a novel Open Sandwich ELISA (OS ELISA) for the detection of 13-(E,E)-HODE. The OS ELISA showed a limit of detection (LOD) of 15.6 nM of 13-(E,E)-HODE and low cross-reactivity with other HODE such as 9-(E,E)-HODE. Another format of the open sandwich ELISA with purified maltose binding protein-fused VL and VH-phage showed a lower LOD of 2.2 nM of 13-(E,E)-HODE, which may be sensitive enough to detect the concentration of 13-(E,E)-HODE in patients' blood samples. This is the first OS ELISA for the detection of lipids, and we believe it also represents the first molecular cloning of anti-HODE antibody genes.