Specific Binding Proteins Research Articles

Effect of the N‐terminal Region of Arabidopsis thaliana LARP6C in RNA Binding Specificity

The La‐related proteins (LARPs) are a superfamily of RNA‐binding proteins that are distinguished by a core RNA binding domain called the “La Module”. This family is highly conserved across eukaryotes and exerts diverse functions in RNA processing and function. The genetic model vascular plant, Arabidopsis thaliana (At), has three paralogs of LARP6, denoted “A”, “B”, and “C”. Of these paralogs, AtLARP6B and AtLARP6C are more closely related to each other due to an N‐terminal sequence motif known to associate with other RNA binding proteins, the “PAM2w” motif. Previous work evaluated the RNA binding activity of the isolated AtLARP6C La Module. Recent work on vertebrate LARP4A suggests that N‐terminal PAM2 motifs are important for higher‐order assembly of regulatory complexes. We have generated a set of recombinant constructs to test the role of both the NTR and PAM2w motif in RNA binding activity. These protein variants are stably expressed and have been highly purified for use in electrophoretic mobility shift assays (EMSAs) to measure RNA binding activity. Preliminary work indicates that binding affinity is not significantly impacted by either the NTR or the PAM2w motif, and current studies are focusing on the role that these regions play in ligand specificity. By characterizing the binding specificity of these AtLARP6C proteins to a panel of RNA ligands, we will gain significant insight into the contribution of this domain and motif to ligand binding.

RNA‐Protein Interactions Involved in Persistence of Cytomegalovirus

Human Cytomegalovirus (HCMV) is a ubiquitous herpes virus and usually asymptomatic among healthy individuals. Congenital HCMV infections are the leading cause of virally induced birth defects and HCMV causes severe disease in immunocompromised patients. Noncoding RNA is a class of RNA molecules that have a wide range of structures and functions which can exhibit cell‐specified specialization and expression. HCMV encodes many ncRNAs that range in size from small (miRNAs) to large stable introns. HCMV encodes several long noncoding RNAs (lncRNAs) of various function. Previous work has shown that HCMV encodes a lncRNA called RNA5.0. The ortholog of RNA5.0 in the MCVM, RNA7.2, has shown to play a role in virus persistence within host salivary glands. However, the molecular interactions/mechanism of this process remains unclear. In order to begin characterizing the molecular function of RNA5.0, we will use the RaPID system to screen for RNA‐protein interactions. The RaPID system recruits a highly active biotin ligase to tag proteins bound on the RNA. Previous work demonstrated that the 3′ end of RNA5.0 is related to the processing and stability of the RNA. There is a hairpin loop that is absolutely required for RNA stability. Several different length fragments spanning the hairpin loop have been cloned into the RaPID system. These constructs will be used to purify RNA binding proteins. Mass spectrometry analysis will be used to identify specific HCMV RNA binding proteins. The identified binding proteins might point to specific pathways involved in viral persistence that could be targeted for potential treatment for CMV.

RanBP3L is a NFAT5 target gene and its deficiency in principal cells promotes epithelial mesenchymal transition through MAPK signaling

Inner medullary kidney cells are exposed to an increasing hyperosmolality altering several intracellular processes. As a key regulator involved in adaption to hyperosmotic environment the transcription factor nuclear factor of activated T cells‐5 (NFAT5) was identified. Its nuclear translocation results in activation of different genes to restore homeostasis. By using primarily cultured rat renal inner medullary collecting duct cells (IMCD) and microarray analysis we identified hundreds of genes that showed differences in expression level in a time‐ and osmolality‐dependent manner. The kidney specific Ran binding protein 3 like (RanBP3L) showed the highest changes in expression level and is located in IMCDs. Within the collecting duct its expression is specific to the principal cells. Since studies describing the renal function are missing, we further characterized the cellular and physiological role of RanBP3L in a renal mouse cell line.We used the murine kidney collecting duct cell line mpKCCD creating a RanBP3L deficient cell clone by using CRISPR/Cas9. To investigate its physiological role the gene expression profiles were analyzed by Next Generation Sequencing. We further analyzed the migration and colony forming capability of the cells and used immunofluorescence to study differences in cell morphology and specific signaling pathways. These experiments were performed under isoosmolar (300 mosmol/kg) and hyperosmolar (600 mosmol/kg) conditions. Since RanBP3L expression is induced by hyperosmolality, the NFAT5 dependent expression of RanBP3L was examined through CRISPR/Cas9 mediated NFAT5 knock out.Loss of RanBP3L under 300 mosmol/kg showed no significant changes in the morphology while under 600 mosmol/kg massive changes were induced resulting in a loss of epithelial structures. This correlates with our functional analysis showing a higher migration and invasiveness capacity. Furthermore RanBP3L deficiency is associated with a massive change in gene expression, indicating a dysregulation of stress associated pathways. Immunofluorescence showed that TGF‐β stimulation in wild type induced a similar phenotype to RanBP3L knock out cells. Downstream analysis of MAPK pathway showed that the key proteins JNK, c‐JUN and Smad2 are deregulated in RanBP3L deficient cells resulting in an epithelial mesenchymal transition (EMT) like phenotype, which is a hallmark in cancer development. This matches with the data from the cancer genome atlas from patients with renal clear cell carcinoma (RCC) where low RanBP3L expression level is associated with shorter overall survival. It seems as if the expression level of RanBP3L in kidney cells is crucial for cell protection. Here we could show that RanBP3L expression is reduced in NFAT5 deficient cells, indicating that RanBP3L is a NFAT5 target gene.Taken together we have shown that RanBP3L is expressed NFAT5 dependent and prevents a MAPK associated EMT in renal cells and should be used as a prognostic marker for patients with RCC. Further experiments, like in‐vivo studies of a conditional RanBP3L knock out mouse will give us more insights to resolve the underlying pathway.Support or Funding Informationgerman research foundation

Custom DNA Microarrays Reveal Diverse Binding Preferences of Proteins and Small Molecules to Thousands of G-Quadruplexes.

Single-stranded DNA (ssDNA) containing four guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their DNA-binding specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ∼15,000 potential G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4-binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding specificity of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition.

Field evaluation of sex pheromones and binding specificity of pheromone binding protein 4 in Tryporyza intacta (Lepidoptera: Crambidae)

The recognition of chemical signal including volatile odorants and pheromones is very important in the olfactory physiological behaviors of insects, such as avoiding predators, seeking food and mating partners. The sugarcane borer, Tryporyza intacta is the most harmful insect in sugarcane region in Southeast Asia and Southern China, however, the study of their molecular biology and physiology was limited. Here we demonstrated that the sex pheromone (E11-16:Ald: Z11-16:Ald = 7:3) were most effective to T. intacta. In addition, compared the traditional rubber lure, a new microsphere formulation lure can optimize the trapping effect and might be widely used in the sugarcane growing area. To obtain a better understanding of the olfactory molecular mechanism of pheromone-based mate recognition system, we have cloned the full-length gene of the TintPBP4 and expressed in Escherichia coli. Our phylogenetic analysis highlighted that the TintPBP4 was highly conserved among diverse species of Lepidoptera. Furthermore, the results of QRT-PCR demonstrated that TintPBP4 transcripts were abundantly expressed in the antennae of T. intacta, especially in the male adults. The fluorescence binding experiments showed the TintPBP4 exhibited strong binding capacities to the sex pheromone components. These results will not only provide more understanding for the functional analysis of olfactory proteins from T. intacta, but also assist in the exploitation and development of sex pheromones in the integrated biological control of this pest.

Modulation of histone modifications and G-quadruplex structures by G-quadruplex-binding proteins

The functions of local conformations of non-B form DNA and RNA, such as the G-quadruplex, are thought to be regulated by their specific binding proteins. They regulate the formation of G-quadruplexes in cells and affect the biological functions of G-quadruplexes. Recent studies reported that G-quadruplexes regulate epigenetics through these G-quadruplex binding proteins. We discuss regulation of histone modifications through G-quadruplex RNA and its binding proteins which modulate the G-quadruplex conformations. G-quadruplex RNA is involved in telomere maintenance and transcription via histone modification. Furthermore, G-quadruplex binding proteins regulate formation and biological functions of G-quadruplexes through regulating their folding or unfolding. In this review, we will focus on the G-quadruplex binding proteins containing RRM and RGG domains.

Analysis of proximal ALOX5 promoter binding proteins by quantitative proteomics.

5-Lipoxygenase (5-LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5-LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter-interacting proteins, obtained by DNA pulldown and label-free quantitative mass spectrometry. Protein preparations from myeloid and B-lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specific ALOX5 promotor binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel-like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G-quadruplex structures. As G-quadruplexes are implicated in gene regulation, spectroscopic and antibody-based methods were used to confirm their presence within the GC-rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form DNA G-quadruplex structures, which may play a functional role in ALOX5 gene regulation.

Aptamer-Based Field-Effect Transistor for Detection of Avian Influenza Virus in Chicken Serum.

Early diagnosis of the highly pathogenic H5N1 avian influenza virus (AIV) is significant for preventing and controlling a global pandemic. However, there is no existing electrical biosensor for detecting biomarkers for AIV in clinically relevant samples such as chicken serum. Herein, we report the first use of an aptamer-functionalized field-effect transistor (FET) as a label-free sensor for AIV detection in chicken serum. A DNA aptamer is employed as a sensitive and selective receptor for hemagglutinin (HA) protein, which is a biomarker for AIVs. This aptamer is immobilized on a gold microelectrode that is connected to the gate of a reusable FET transducer. The specific binding of the target protein results in a change in the surface potential, which generates a signal response of the FET transducer. We hypothesize that a conformational change in the DNA aptamer upon specific binding of HA protein may alter the surface potential. The signal of the aptamer-based FET biosensor increased linearly with the increase in the logarithm of HA protein concentration in a dynamic range of 10 pM to 10 nM with a detection limit of 5.9 pM. The selectivity of the biosensor for HA protein was confirmed by employing relevant interfering proteins. The proposed biosensor was successfully applied to the selective detection of HA protein in a chicken serum sample. Owing to its simple and low-cost architecture, portability, and sensitivity, the aptamer-based FET biosensor has potential as a point-of-care diagnosis of H5N1 AIVs in clinical samples.

Interaction of a Trichinella spiralis cathepsin B with enterocytes promotes the larval intrusion into the cells

Cathepsin B is one member of cysteine protease family and widely distributed in organisms, it plays an important function in parasite penetrating, migrating, molting and immune escaping. The aim of this work was to investigate whether exist interaction between a Trichinella spiralis cathepsin B (TsCB) and mouse intestinal epithelium cells (IECs), and its influence in the process of larva cell invasion. The results of ELISA, indirect immunofluorescence assay (IIFA), confocal microscopy and Far western blotting showed that there was a strong specific binding of rTsCB and IEC proteins, and the binding positions were located in cytoplasm and nuclei of IECs. The results of the in vitro larva penetration test revealed that rTsCB facilitated the larva invasion of IECs, whereas anti-rTsCB antibodies impeded partially the larva intrusion of enterocytes, this promotive or inhibitory roles were dose-dependent of rTsCB or anti-rTsCB antibodies. Silencing TsCB by siRNA mediated RNA interference reduced the TsCB expression in T. spiralis larvae, and markedly inhibited the larva penetration of enterocytes. The results indicated that TsCB binding to IECs promoted larva penetration of host's enteral epithelia, and it is a promising molecular target against intestinal invasive stages of T. spiralis.

Pervasive functional translation of noncanonical human open reading frames.

Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.

Association of persistent wild-type measles virus RNA with long-term humoral immunity in rhesus macaques.

Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for 6 months after infection with wild-type measles virus (MeV). Infection caused viremia and rash, with clearance of infectious virus by day 14. MeV RNA persisted in PBMCs for 30-90 days and in lymphoid tissue for 6 months most often in B cells but was rarely detected in BM. Antibody with neutralizing activity and binding specificity for MeV nucleocapsid (N), hemagglutinin (H), and fusion proteins appeared with the rash and avidity matured over 3-4 months. Lymph nodes had increasing numbers of MeV-specific antibody-secreting cells (ASCs) and germinal centers with late hyalinization. ASCs appeared in circulation with the rash and continued to appear along with peripheral T follicular helper cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody against both H and N, with more H-specific ASCs in BM. During days 14-21, 20- to 100-fold more total ASCs than MeV-specific ASCs appeared in circulation, suggesting mobilization of preexisting ASCs. Therefore, persistence of MeV RNA in lymphoid tissue was accompanied by continued germinal center formation, ASC production, avidity maturation, and accumulation of H-specific ASCs in BM to sustain neutralizing antibody and protective immunity.

Developmental and sexual divergence in the olfactory system of the marine insect Clunio marinus

An animal’s fitness strongly depends on successful feeding, avoidance of predators and reproduction. All of these behaviours commonly involve chemosensation. As a consequence, when species’ ecological niches and life histories differ, their chemosensory abilities need to be adapted accordingly. The intertidal insect Clunio marinus (Diptera: Chironomidae) has tuned its olfactory system to two highly divergent niches. The long-lived larvae forage in a marine environment. During the few hours of terrestrial adult life, males have to find the female pupae floating on the water surface, free the cryptic females from their pupal skin, copulate and carry the females to the oviposition sites. In order to explore the possibility for divergent olfactory adaptations within the same species, we investigated the chemosensory system of C. marinus larvae, adult males and adult females at the morphological and molecular level. The larvae have a well-developed olfactory system, but olfactory gene expression only partially overlaps with that of adults, likely reflecting their marine vs. terrestrial lifestyles. The olfactory system of the short-lived adults is simple, displaying no glomeruli in the antennal lobes. There is strong sexual dimorphism, the female olfactory system being particularly reduced in terms of number of antennal annuli and sensilla, olfactory brain centre size and gene expression. We found hints for a pheromone detection system in males, including large trichoid sensilla and expression of specific olfactory receptors and odorant binding proteins. Taken together, this makes C. marinus an excellent model to study within-species evolution and adaptation of chemosensory systems.

Molecular Dynamics Simulations of a Chimeric Androgen Receptor Protein (SPARKI) Confirm the Importance of the Dimerization Domain on DNA Binding Specificity.

The DNA binding domains of Androgen/Glucocorticoid receptors (AR/GR), members of class I steroid receptors, bind as a homo-dimer to a cis-regulatory element. These response elements are arranged as inverted repeat (IR) of hexamer “AGAACA”, separated with a 3 base pairs spacer. DNA binding domains of the Androgen receptor, AR-DBDs, in addition, selectively recognize a direct-like repeat (DR) arrangement of this hexamer. A chimeric AR protein, termed SPARKI, in which the second zinc-binding motif of AR is swapped with that of GR, however, fails to recognize DR-like elements. By molecular dynamic simulations, we identify how the DNA binding domains of the wild type AR/GR, and also the chimeric SPARKI model, distinctly interact with both IR and DR response elements. AR binds more strongly to DR than GR binds to IR elements. A SPARKI model built from the structure of the AR (SPARKI-AR) shows significantly fewer hydrogen bond interactions in complex with a DR sequence than with an IR sequence. Moreover, a SPARKI model based on the structure of the GR (SPARKI-GR) shows a considerable distortion in its dimerization domain when complexed to a DR-DNA whereas it remains in a stable conformation in a complex with an IR-DNA. The diminished interaction of SPARKI-AR with and the instability of SPARKI-GR on DR response elements agree with SPARKI's lack of affinity for these sequences. The more GR-like binding specificity of the chimeric SPARKI protein is further emphasized by both SPARKI models binding even more strongly to IR elements than observed for the DNA binding domain of the GR.

A Colorimetric Artificial Olfactory System for Airborne Improvised Explosive Identification

The detection of ultralow or nonvolatile target analytes remains a significant challenge for artificial olfactory systems even after decades of development, which severely limits their widespread application. To overcome this challenge, an artificial olfactory system based on a colorimetric hydrogel array is constructed for the first time as a universal representative. As an effective extension of conventional artificial olfactory systems that integrates the merits of its predecessors, the proposed system accurately mimics olfactory mucosa and specific odorant binding proteins using hydrogels endowed with specific colorimetric reagents for the detection of hypochlorite, chlorate, perchlorate, urea, and nitrate. Therefore, the proposed system is capable of detecting and discriminating between these five airborne improvised explosive microparticulates with a detection limit as low as 39.4 pg. Additionally, the system demonstrates good reusability over ten cycles, rapid response time of ≈0.2 s, and excellent discrimination properties, despite significant variation. This proof-of-concept study on colorimetric artificial olfactory systems yields a novel strategy for the direct and discriminative detection of nonvolatile airborne microparticulates.

Zinc Transporter-3 Knockout Mice Demonstrate Age-Dependent Alterations in the Metalloproteome

Metals are critical cellular elements that are involved in a variety of cellular processes, with recent literature demonstrating that zinc, and the synaptic zinc transporter (ZnT3), are specifically involved in learning and memory and may also be key players in age-related neurodegenerative disorders such as Alzheimer’s disease. Whilst the cellular content and location of metals is critical, recent data has demonstrated that the metalation state of proteins is a determinant of protein function and potential toxicity. As we have previously reported that ZnT3 knockout (KO) mice have deficits in total zinc levels at both 3 and 6 months of age, we were interested in whether there might be changes in the metalloproteomic profile in these animals. To do this, we utilised size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and examined hippocampal homogenates from ZnT3 KO and age-matched wild-type mice at 3, 6 and 18 months of age. Our data suggest that there are alterations in specific metal binding proteins, for zinc, copper and iron all being modulated in the ZnT3 KO mice compared to wild-type (WT). These data suggest that ZnT3 KO mice may have impairments in the levels or localisation of multiple transition metals, and that copper- and iron-dependent cellular pathways may also be impacted in these mice.

Study of the noncovalent interactions between phenolic acid and lysozyme by cold spray ionization mass spectrometry (CSI-MS), multi-spectroscopic and molecular docking approaches

Elucidating the recognition mechanisms of the noncovalent interactions between pharmaceutical molecules and proteins is important for understanding drug delivery in vivo, and for the further rapid screening of clinical drug candidates and biomarkers. In this work, a strategy based on cold spray ionization mass spectrometry (CSI-MS), combined with fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and molecular docking methods, was developed and applied to the study of the noncovalent interactions between phenolic acid and lysozyme (Lys). Based on the real characterization of noncovalent complex, the detailed binding parameters, as well as the protein conformational changes and specific binding sites could be obtained. CSI-MS and tandem mass spectrometry (MS/MS) technique were used to investigate the phenolic acid-Lys complexes and the structure-affinity relationship, and to assess their structural composition and gas phase stability. The binding affinity was obtained by direct and indirect MS methods. The fluorescence spectra showed that the intrinsic fluorescence quenching of Lys in solution was a static quenching mechanism caused by complex formation, which supported the MS results. The CD and FTIR spectra revealed that phenolic acid changed the secondary structure of Lys and increased the α-helix content, indicating an increase in the tryptophan (W) hydrophobicity near the protein binding site resulting in a conformational alteration of the protein. In addition, molecular docking studies were performed to investigate the binding sites and binding modes of phenolic acid on Lys. This strategy can more comprehensively and truly characterize the noncovalent interactions and can guide further research on the interactions of phenolic acid with other proteins.

Dynamical control of denaturation bubble nucleation in supercoiled DNA minicircles

We examine the behavior of supercoiled DNA minicircles containing between 200 and 400 base-pairs, also named microDNA, in which supercoiling favors thermally assisted DNA denaturation bubbles of nanometer size and controls their lifetime. Mesoscopic modeling and accelerated dynamics simulations allow us to overcome the limitations of atomistic simulations encountered in such systems, and offer detailed insight into the thermodynamic and dynamical properties associated with the nucleation and closure mechanisms of long-lived thermally assisted denaturation bubbles which do not stem from bending- or torque-driven stress. Suitable tuning of the degree of supercoiling and size of specifically designed microDNA is observed to lead to the control of opening characteristic times in the millisecond range, and closure characteristic times ranging over well distinct timescales, from microseconds to several minutes. We discuss how our results can be seen as a dynamical bandwidth which might enhance selectivity for specific DNA binding proteins.

Specific binding of DnaA to the DnaA box motif in the cyanobacterium Synechococcus elongatus PCC 7942.

In bacterial DNA replication, the initiator protein DnaA binds to the multiple DnaA box sequences located at oriC to facilitate the unwinding of duplex DNA strands. The cyanobacterium Synechococcus elongatus PCC 7942, which contains multiple chromosomal copies per cell, has DnaA box (like sequences around the oriC region, which is located upstream of dnaN. We previously observed the binding of DnaA around the oriC region; however, the DNA-binding specificity of DnaA to DnaA box sequences has not been examined. Here, we analyzed the binding specificity of DnaA protein to the DnaA box in S. elongatus by using bio-layer interferometry (BLI), a method for monitoring intermolecular interactions. We observed that recombinant DnaA protein recognized specifically the DnaA box sequence TTTTCCACA in vitro. In addition, DNA binding activity was significantly increased by R328H mutation of DnaA. This is the first report to characterize DnaA binding to the DnaA box sequence in cyanobacteria.

Colorectal Carcinoma Presenting as Ovarian Metastasis: A Case Report

There is a common occurrence of colorectal adenocarcinoma metastasis to the ovary and in rare circumstances, these cases may present clinically as primary ovarian cancer and histologically as primary endometrioid carcinoma of the ovary. Approximately 3.6% to 7.4% of patients with colon cancer have ovarian metastasis at the time of initial presentation, of which, 45% are mistaken for primary ovarian tumors. The author reports the case of a 60-year-old female presenting with fatigue and pelvic pain secondary to a pelvic mass. Computed tomography (CT) revealed a lobulated, heterogeneous pelvic mass measuring 11.1 × 10.5 × 9.8 cm, and ultrasound (US) showed complex 13.8 × 8.2 × 12.3 cm mass posterior to the uterus. Upon examination of the specimen following debulking procedure, endometrioid carcinoma of the ovaries was initially considered. However, immunohistochemical stains were performed and showed malignant cells positive for cytokeratin (CK) 20, caudal type homeobox (CDX) 2, and specific AT-rich sequence binding (SATB) protein 2, consistent with metastatic colorectal carcinoma. This report highlights the diagnostic challenges arising with differentiation between primary endometrioid ovarian carcinoma and metastatic colorectal adenocarcinoma to the ovary and the potential clinical consequences of misdiagnosis.

Responses to Higher Concentration Lecithin of Apoptosis Relevant Factors and Detection of Specific Lecithin Binding Proteins by AC-MS assay

Lecithin in mixed micelles attenuates cytotoxicity by interacting with proteins or other cell inclusions. The reaction was then captured via magnetic beads binding approach. This research proposal was designed to discover the impacts of lecithin on apoptosis relevant factors (including caspase 3/8/9 and PARP) by Western Blot and specific protein binding mechanism of lecithin detoxification pathway, using caco-2 cell model. Preliminary investigation of the properties of the proteins was conducted by MS (Mass Spectrometry) couplet to AC (Affinity Chromatography) assay. Further research of particular proteins was to identify the size and peptide sequence. This study also provided solid evidence for the previous research result, which was lecithin reduce cytotoxicity induced by SDC contained mixed micelles.