Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Synthesis of Submicron-Sized Spherical Silica-Coated Iron Nickel Particles with Adjustable Shell Thickness via Swirler Connector-Assisted Spray Pyrolysis.

Silica-coated iron nickel (FeNi@SiO2) particles have attracted significant attention because of their potential applications in electronic devices. In this work, submicron-sized spherical FeNi@SiO2 particles with precisely controllable shell thickness were successfully synthesized for the first time using a swirler connector-assisted spray pyrolysis system, comprising a preheater, specific connector, and main heater. The results indicated that the thickness of the SiO2 shell can be tuned from 3 to 23 nm by adjusting the parameter conditions (i.e., preheater temperature, SiO2 supplied amount). Furthermore, our fabrication method consistently yielded a high coating ratio of more than 94%, indicating an excellent quality of the synthesized particles. Especially, to gain an in-depth understanding of the particle formation process of the FeNi@SiO2 particles, a plausible mechanism was also investigated. These findings highlight the importance of controlling the preheater and SiO2 supplied amount to obtain FeNi@SiO2 particles with desirable morphology and high coating quality.

Mechanisms that can cause population decline under heavily skewed male-biased adult sex ratios.

While adult sex ratio (ASR) is a crucial component for population management, there is still a limited understanding of how its fluctuation affects population dynamics. To demonstrate mechanisms that hinder population growth under a biased ASR, we examined changes in reproductive success with ASR using a decapod crustacean exposed to female-selective harvesting. We examined the effect of ASR on the spawning success of females. A laboratory experiment showed that the number of eggs carried by females decreased as the proportion of males in the mating groups increased. Although the same result was not observed in data collected over 25 years in the wild, the negative effect of ASR was suggested when success in carrying eggs was considered as a spawning success. These results indicate that a surplus of males results in females failing to carry eggs, probably due to sexual coercion, and the negative effect of ASR can be detected at the population level only when the bias increases because failure in spawning success occurs in part of population. We experimentally examined how male-biased sex ratios affected the maintenance of genetic diversity in a population. The diversity of paternity in a clutch increased with the number of candidate fathers. However, over 50% of a clutch was fertilised by a single male regardless of the sex ratio, and the degree of diversity was less than half of the highest diversity expected in each mating group. We also experimentally examined the mating ability of males during the breeding season. The experiment showed that multiple mating by males could not compensate for the risk that their genotypes would be lost when multiple males competed for one female. These results suggest that a male-biased ASR could trigger a decline of genetic diversity in a population. We show that ASR skewed by female-selective harvesting decreases reproductive success not only of males that have few mating opportunities but also of females. We discuss that we may still underestimate the significance of ASR on population persistence due to the difficulty of revealing the effect of ASR.

Mycobacterium abscessus subsp. abscessus empyema complicated with subcutaneous abscess.

There have been no case reports of thoracic subcutaneous abscess after surgery for Mycobacterium abscessus complex associated empyema. We herein report a case of Mycobacterium abscessus subsp. abscessus (M. abscessus subsp. abscessus) induced subcutaneous abscesses following surgical treatment for concurrent M. abscessus subsp. abscessus -associated empyema and pneumothorax. A 75-year-old woman had M. abscessus subsp. abscessus -associated empyema and pneumothorax. She underwent surgical treatment of decortication and fistulectomy and suffered from M. abscessus subsp. abscessus -associated subcutaneous abscesses after thoracentesis/drainage. A multidisciplinary approach combined with surgical care, thermal therapy, and multidrug chemotherapy contributed to a successful result. An early multidisciplinary approach is believed to be important in cases of M. abscessus subsp. abscessus -associated empyema and subcutaneous abscess.

[Percutaneous Transluminal Sinus Stenting for Sigmoid Sinus Stenosis Associated with Transverse-Sigmoid Sinus Dural Arteriovenous Fistula:A Case Report].

We report a case of transverse sinus-sigmoid sinus dural arteriovenous fistula(T-S dAVF)with venous flow congestion, which was accompanied by sigmoid sinus stenosis and treated with percutaneous transluminal sinus stenting. A 76-year-old woman presented with dementia and disturbance of consciousness. Magnetic resonance imaging(MRI)on admission revealed subcortical edema in the left occipital lobe and angiography demonstrated a left T-S dAVF with right transverse sinus occlusion and sigmoid sinus stenosis. Hemodynamics of the shunt flow from several feeders demonstrated retrograde flow to the straight sinus and superior sagittal sinus, and antegrade flow into the left internal jugular vein. These hemodynamics caused cortical venous congestion and intracranial venous hypertension. We performed percutaneous transluminal sinus stenting for left sigmoid sinus stenosis. Immediately after stent placement, retrograde shunt flow to the straight sinus and superior straight sinus dramatically disappeared and cortical venous congestion improved. Follow-up angiography 1 year after treatment showed neither new development of T-S dAVF nor re-stenosis of the stent in the left sigmoid sinus, although some shunt flow remained. Percutaneous transluminal sinus stenting for sinus stenosis associated with dAVF appears effective to improve venous congestion and intracranial venous hypertension.

Elevated Serum Anti-GM-CSF Antibodies before the Onset of Autoimmune Pulmonary Alveolar Proteinosis in a Patient with Sarcoidosis and Systemic Sclerosis.

Pulmonary alveolar proteinosis (PAP) is characterized by the accumulation of periodic acid-schiff stain-positive lipoproteinaceous materials in the alveolar space due to impaired surfactant clearance by alveolar macrophage. Autoimmune PAP is the most common form of PAP, but rarely accompanies collagen disease or sarcoidosis. We report here a rare case of autoimmune PAP preceded by systemic sclerosis and sarcoidosis. A 64-year-old woman was admitted to our hospital for blurred vision, muscle weakness of extremities, Raynaud's phenomenon, and exertional dyspnea. We diagnosed her as having systemic sclerosis complicated with sarcoidosis. Chest computed tomography (CT) and transbronchial lung biopsy showed the findings of pulmonary fibrosis without PAP. We treated her with corticosteroid and intravenous cyclophosphamide therapy, followed by tacrolimus therapy. Thereafter, her symptoms improved except for exertional dyspnea, and she began to complain of productive cough thirteen months after corticosteroid and immunosuppressant therapy. On the second admission, a chest CT scan detected the emergence of crazy-paving pattern in bilateral upper lobes. Bronchoalveolar lavage (BAL) fluid with milky appearance and a lung biopsy specimen revealed acellular periodic acid-schiff stain-positive bodies. The serum titer of anti-granulocyte macrophage colony stimulating factor (GM-CSF) antibodies was elevated on first admission and remained high on second admission. We thus diagnosed her as having autoimmune PAP. Reducing the dose of immunosuppressive agents and repeating the segmental BAL resulted in the improvement of her symptoms and radiological findings. Immunosuppressant therapy may trigger the onset of autoimmune PAP in a subset of patients with systemic sclerosis and/or sarcoidosis.

[A Case of Tentorial Dural Arteriovenous Fistula Treated with Transvenous Embolization Using NBCA].

We report a case of tentorial dural arteriovenous fistula(dAVF)treated with transarterial and transvenous embolization using n-butyl-2-cyanoacrylate(NBCA). A 70-year-old man presented with dysarthria and trunk ataxia. Computed tomography(CT)on admission revealed right cerebellar hemorrhage. Right external carotid angiography demonstrated a tentorial dAVF fed by the marginal tentorial artery, petrosquamous branch of the middle meningeal artery, ascending pharyngeal artery, and artery of foramen rotundum. Right internal carotid angiography demonstrated a shunt fed by the meningohypophyseal trunk. The draining vein was the right basal vein with a varix, which drained into the straight sinus. Thin-slice axial images on magnetic resonance angiography demonstrated a shunt point located on the right tentorial incisura. The lesion was diagnosed as Cognard type IV tentorial dAVF. It was initially treated with transarterial embolization using 25% NBCA, which was injected into the marginal tentorial artery and the petrosquamous branch of the middle meningeal artery. However, owing to partial persistence of the shunt after the procedure, transvenous embolization using NBCA was performed. A microcatheter was navigated through the straight sinus into the basal vein, and a balloon catheter was also navigated to the confluence of the straight sinus and the basal vein to interrupt blood flow and prevent the NBCA from flowing back to the sinus. 80% NBCA was injected into the draining vein near the shunt point. Angiography performed immediately after the procedure revealed complete occlusion of the shunt, and postoperative CT showed no evidence of hemorrhage. Transvenous embolization of tentorial dAVF can be an effective method if a microcatheter can be safely advanced close to the shunt point.

[Biomarkers of Leukemia ~Description of the Practical Use and Operation in the Clinical Laboratory~]-.

Genetic testing of hematological malignancy is-.indispensable to categorize and diagnose leukemia. The quantitation of fusion gene mRNA built up by chromosomal translocation including BCR-ABL1 (major, minor), RUNX1-RUNX1T1, and PML-RARA and detection of the JAK2 (V617F) mutant gene are performed by our- selves in our hospital. Efficient, practical use is necessary because the number of medical technologists is limited and numbers of tests are increasing annually. The detection of leukemic cells is important in hematological tests. In addition, experienced medical technologists can predict the existence of fusion mutant genes. In this report, we introduce our experience regarding the practical use and operation of biomarkers for leukemia. Medical technologists take advantage of peripheral blood tests for screening, such as the complete blood count (CBC), hemogram, fibrin and fibrinogen degradation products (FDP), and the quantitation of fusion gene mRNA, which offers a definitive diagnosis including BCR-ABL1, RUNX1-RUNX1T1, and PML-RARA, and genetic tests are performed efficiently. [Review].

Multianalyte Conventional Reference Material (MacRM): A Useful Tool for Nationwide Standardization of Laboratory Measurements for Medical Care-A Model Study in Japan.

BACKGROUND: The Japanese Committee for Clinical Laboratory Standards (JCCLS) has developed a multianalyte conventional reference material (MacRM) for nationwide standardization of laboratory measurements. METHODS: To prepare the MacRM, pooled sera were obtained from healthy Japanese individuals. Target values of the pooled sera for 30 analytes were assigned on the basis of the measurement results of 45 certified clinical laboratories whose calibration was verified by measuring certified reference materials (CRMs) provided by the National Institute of Standards and Technology, the Institute for Reference Materials and Measurements, and JCCLS. Commutability of MacRM was assessed by comparison with results for 150 individual inpatients at Fukuoka University Chikushi Hospital. Survey samples were prepared by essentially the same method for MacRM but without target values. The survey samples were used to assess agreement among 165 laboratories that used various assay kits and platforms calibrated with the MacRM. RESULTS: The commutability of MacRM was confirmed for 30 analytes with sera from 150 individual patients. The imprecision (CV) of measurements of survey samples (high and low concentrations) among the 165 laboratories was 0.4%-10.0%. Twenty-six of 30 analytes were within the goals for interinstitutional allowable bias. An aliquot of MacRM stored frozen at -80 °C remained stable for ≥4 years. CONCLUSIONS: The MacRM was successfully applied as a calibrator to achieve nationwide standardization for 30 analytes measured by 165 laboratories that used various methods from different manufacturers.

A novel type 3 secretion system effector, YspI of Yersinia enterocolitica, induces cell paralysis by reducing total focal adhesion kinase.

Some of the world's most important diseases are caused by bacterial pathogens that deliver toxic effector proteins directly into eukaryotic cells using type III secretion systems. The myriad of pathological outcomes caused by these pathogens is determined, in part, by the manipulation of host cell physiology due to the specific activities of individual effectors among the unique suite each pathogen employs. YspI was found to be an effector, delivered by Yersinia enterocolitica Biovar 1B, that inhibits host cell motility. The action of YspI comes about through its specific interaction with focal adhesion kinase, FAK, which is a fulcrum of focal adhesion complexes for controlling cellular motility. The interaction was defined by a specific domain of YspI that bound to the FAK kinase domain. Further examination revealed that YspI-FAK interaction leads to a reduction of FAK steady-state levels without altering its phosphorylation state. This collection of observations and results showed YspI displays unique functionality by targeting the key regulator of focal adhesion complexes to inhibit cellular movement.

Corticosteroid Therapy for a Patient with Relapsing Polychondritis Complicated by IgG4-Related Disease.

Relapsing polychondritis (RP) is a rare systemic disorder characterized by recurrent, widespread chondritis of the auricular, nasal, and tracheal cartilages. IgG4-related disease (IgG4-RD) is a systemic immune-mediated disease characterized by the infiltration of IgG4-bearing plasma cells into systemic organs. Although 25% to 35% of patients with RP have a concurrent autoimmune disease, coexistence of RP and IgG4-RD is rare. We herein report a case of RP complicated by IgG4-RD. A 63-year-old man developed recurrent bilateral ear pain and swelling, recurrent blurred and decreased vision, and migratory multiple joint pain, sequentially within one year. Fourteen months after the first symptom, he experienced dry cough and dyspnea with exertion. A computed tomography (CT) scan detected interstitial pneumonia, swelling of bilateral submandibular glands, bilateral hilar and mediastinal lymphadenopathy, and several nodules in bilateral kidneys. His serum levels of IgG and IgG4 were elevated. The biopsy specimen of auricular cartilage showed infiltrations of inflammatory cells and fibrosis consistent with RP. The IgG4-positive cells were not observed in auricular cartilage. The patient met the diagnostic criteria of RP, including bilateral auricular chondritis, conjunctivitis, iritis and polyarthritis. The biopsy specimens of lung and kidney revealed the significant infiltrations of IgG4-positive plasma cells and fibrosis. We also diagnosed him as having IgG4-RD, affecting bilateral submandibular glands, hilar and mediastinal lymph nodes, lungs, and kidneys. Thus, RP preceded the onset of IgG4-RD. Corticosteroid therapy improved the symptoms and CT scan findings. In conclusion, RP and IgG4-RD do coexist; however, the pathogenesis of their coexistence is unknown.

[Chairmen's Introductory Remarks].

At the 62nd Annual Meeting of the Japanese Society of Laboratory Medicine Scientific Sessions, a "promotion of globalization in clinical laboratory area" symposium of the Japanese Society of Laboratory Medicine (JSLM) and the Japanese Association of Medical Technologists (JAMT) was held. Studying abroad as well as the overseas dispatch of biomedical laboratory scientists have increased in re- cent years. However, there are still a number of problems. Both the JSLM and JAMT are focusing on in- ternational activities. For example, the JSLM has the International Committee, and the JAMT has set up a specially appointed Executive Director of International Affairs. Moreover, international standards, facility certification, and clinical trials are being established at the clinical laboratory. In other words, there is a growing need for the third-party evaluation of such international certification. The promotion of globaliza- tion in the clinical laboratory area is essential, and human resource development to nurture an international community in the future is important. However, the most important thing is that many biomedical laborato- ry scientists participate in international conferences. [Review].

[Harmonization of TSH Measurements.]

The measured concentration of thyroid stimulating hormone (TSH) differs depending on the reagents used. Harmonization of TSH is crucial because the decision limits are described in current clinical practice guide- lines as absolute values, e.g. 2.5 mIU/L in early pregnancy. In this study, we tried to harmonize the report- ed concentrations of TSH using the all-procedure trimmed mean. TSH was measured in 146 serum samples, with values ranging from 0.01 to 18.8 mIU/L, using 4 immunoassays. The concentration of TSH was highest with E test TOSOH and lowest with LUMIPULSE. The concentrations with each reagent were recalculated with the following formulas: E test TOSOH 0.855x-0.014; ECLusys 0.993x+0.079; ARCHITECT 1.041x- 0.010; and LUMIPULSE 1.096x-0.015. Recalculation eliminated the between-assay discrepancy. These formulas may be used until harmonization of TSH is achieved by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC).

The role of the non-covalent ß-ionone-ring binding site in rhodopsin: historical and physiological perspective.

Bleached rhodopsin regenerates by way of the Schiff base formation between the 11-cis retinal and opsin. Recovery of human vision from light adapted states follows biphasic kinetics and each adaptive phase is assigned to two distinct classes of visual pigments in cones and rods, respectively, suggesting that the speed of Schiff base formation differs between iodopsin and rhodopsin. Matsumoto and Yoshizawa predicted the existence of a ß-ionone ring-binding site in rhodopsin, which has been proven by structural studies. They postulated that rhodopsin regeneration starts with a non-covalent binding of the ß-ionone ring moiety of 11-cis-retinal, followed by the Schiff base formation. Recent physiological investigation revealed that non-covalent occupation of the ß-ionone ring binding site transiently activates the visual transduction cascade in the dark. In order to understand the role of non-covalent binding of 11-cis-retinal to opsin during regeneration, we studied the kinetics of rhodopsin regeneration from opsin and 11-cis-retinal and found that the Schiff base formation is accelerated ∼10(7) times compared to that between retinal and free amine. According to Cordes and Jencks, Schiff base formation in solution exhibits a bell-shaped pH dependence. However, we discovered that the rhodopsin formation is independent of pH over a wide pH range, suggesting that aqueous solvents do not have access to the Schiff base milieu during its formation. According to Hecht et al. the regeneration of iodopsin must be significantly faster than that of rhodopsin. Does this suggest that the Schiff base formation in iodopsin is favored due to its structural architecture? The iodopsin structure once solved would answer such a question as how molecular fine-tuning of retinal proteins realizes their dark adaptive functions. In contrast, bacteriorhodopsin does not require occupancy of a distinct ß-ionone ring-binding site, enabling an aldehyde without the cyclohexene ring to form a pigment. Studies of regeneration reaction of other retinal proteins, which are scarcely available, would clarify the molecular structure-phenotype relationships and their physiological roles.

[A Case of Percutaneous Transluminal Angioplasty and Stenting for Dacron Bypass Graft Stenosis with Cerebral Infarction].

A 62-year-old man presented to the emergency room with mild dysarthria and right motor weakness. The patient was diagnosed with aortic dissection (DeBakey type III) in the cardiovascular department of our institution two years ago and was then treated with left carotid-bilateral subclavian bypass with collagen-seated Dacron graft followed by thoracic endovascular aortic repair (TEVER) with stent-graft placement. Magnetic resonance imaging on admission showed cerebral infarction with left proximal middle cerebral artery occlusion in the left cerebral hemisphere. Three-dimensional computed tomography angiography (3D-CTA) demonstrated a stenotic lesion at the anastomosis of the right subclavian artery and the bypass graft. It also showed the partial left common carotid artery, suggestive of an endoleak in the thoracic stent graft. The patient was diagnosed with artery-to-artery embolism due to bypass graft stenosis or endoleak in the thoracic stent graft and was treated with conservative therapy. He gradually recovered from the neurological deficit and underwent endovascular angioplasty with a balloon-expandable stent for bypass graft stenosis by using the distal balloon protection method and the left proximal common carotid artery occlusion with coils 1 month later. One-year follow-up 3D-CTA showed good patency of the stent in the bypass graft. No recurrence of cerebral infarction was observed during the postoperative course.

[KRAS Genetic Mutation Analysis Using the Clinical FFPE Samples of Colorectal Carcinomas: Comparative Study among 5 Methods].

Targeting of epidermal growth factor receptor (EGFR) with monoclonal antibodies such as cetuximab or panitumumab inhibits the activation of downstream signaling molecules of EGFR. Since EGFR inhibitor therapy has been approved for the treatment of colorectal carcinomas in patients with tumors lacking KRAS mutations, the detection of KRAS mutation is indispensable before starting therapy. Although several laboratory procedures have been developed to detect KRAS mutations, few comparative studies of the sensitivity of mutation detection have been performed for such procedures. Here, we compared the levels of detection in 5 commercial KRAS mutation detection methods using both standard DNA samples and formalin-fixed clinical samples obtained during surgery. Scorpion-ARMS assay has been reported as the most sensitive and we compared 4 other methods with Scorpion. Both PCR-rSSO assay and F-PHFA assay showed the highest concordance. As for the detection sensitivity, there were variations between clinical samples apparently due to sample quality or assay principles and methods. It is thus suggested that routine validation is required using samples prepared in each lab and the choice of assay may depend on various factors, such as lab environment, actual needs of physicians and patients, and the quality of the formalin-fixed specimens.

[Outcomes of Infection Control Team Inspections at the Dental Hospital, Tokyo Medical and Dental University].

In the Dental Hospital, Tokyo Medical and Dental University, an infection control team (ICT) has been formed to inspect each diagnosis department of clinics and wards in order to identify problems regarding nosocomial infection control. In this study, we analyzed the inspection reports and highlighted the following serious problems: 1) inadequate hygienic hand-washing for out- and in-patient treatment, 2) incomplete wearing of personal protective equipment (PPE) by dental health care workers, 3) necessity of environmental improvement in the clinics, and 4) cross-infection risk induced by. the continuous use of treatment devices without appropriate disinfection. The ICT provided feedback to the inspected departments, suggesting solutions to problems regarding nosocomial infection control. In order to enhance infection control in our hospital, dental healthcare practitioners must make further efforts on nosocomial infection control and prevention, and act according to their position by continuously educating students and enlightening hospital staff about the importance of infection control.

Efficient extraction of proteins from formalin-fixed paraffin-embedded tissues requires higher concentration of tris(hydroxymethyl)aminomethane.

BACKGROUND: Numerous formaldehyde-fixed and paraffin-embedded clinical tissues have been created in the past decades and stored in pathological depositories at hospitals as well as in clinical laboratories worldwide. In addition to the archived tissues, formaldehyde-fixation is also mandatory for preparing proteomics samples from diseased patients or animal models in order to inactivate contagious agents. Protein extraction from formaldehyde-fixed tissues is hampered by the Schiff base formation between the amino groups of proteins and formaldehyde. Although achievement of the highest extraction efficiency of proteins from the formaldehyde-fixed tissues is essential for obtaining maximum proteomics information, no attention has been paid to the concentration dependence of tris(hydroxymethyl)aminomethane on the extraction efficacy. We suspected that the concentration of tris(hydroxymethyl)aminomethane affects the protein extraction efficiency because of its property as a primary amine that reverses the Schiff base formation between the primary amines of proteins and formaldehyde. Thus we pursued optimization of the component and protocol of protein extraction buffer to achieve better extraction efficiency of proteins from formaldehyde-fixed and paraffin-embedded tissues. RESULTS: In order to simulate protein extraction from diseased tissues we made formaldehyde-fixed and paraffin-embedded samples from mouse liver slices and investigated the protein extraction efficiency and speed by changing the concentration of the protein extraction buffer component tris(hydroxymethyl)aminomethane under various extraction conditions. We find, as expected, that tris(hydroxymethyl)aminomethane significantly affects the performance of protein extraction from the formaldehyde-fixed and paraffin-embedded samples both in the extraction yield and in the extraction speed. CONCLUSIONS: We recommend the concentration of tris(hydroxymethyl)aminomethane in protein extraction buffer to be higher than 300 mM when extraction is conducted for 90 min at 90°C to achieve the most efficient protein extraction in a shorter time. The information will be essential for performing the most efficient protein extraction from formaldehyde-fixed and paraffin-embedded tissue samples for proteomics analysis.

SHetA2 interference with mortalin binding to p66shc and p53 identified using drug-conjugated magnetic microspheres.

SHetA2 is a small molecule flexible heteroarotinoid (Flex-Het) with promising cancer prevention and therapeutic activity. Extensive preclinical testing documented lack of SHetA2 toxicity at doses 25 to 150 fold above effective doses. Knowledge of the SHetA2 molecular target(s) that mediate(s) the mechanism of SHetA2 action is critical to appropriate design of clinical trials and improved analogs. The aim of this study was to develop a method to identify SHetA2 binding proteins in cancer cells. A known metabolite of SHetA2 that has a hydroxyl group available for attachment was synthesized and conjugated to a linker for attachment to a magnetic microsphere. SHetA2-conjugated magnetic microspheres and unconjugated magnetic microspheres were separately incubated with aliquots of a whole cell protein extract from the A2780 human ovarian cancer cell line. After washing away non-specifically bound proteins with the protein extraction buffer, SHetA2-binding proteins were eluted with an excess of free SHetA2. In two independent experiments, an SDS gel band of about 72 kDa was present at differential levels in wells of eluent from SHetA2-microspheres in comparison to wells of eluent from unconjugated microspheres. Mass spectrometry analysis of the bands (QStar) and straight eluents (Orbitrap) identified mortalin (HSPA9) to be present in the eluent from SHetA2-microspheres and not in eluent from unconjugated microspheres. Co-immunoprecipitation experiments demonstrated that SHetA2 interfered with mortalin binding to p53 and p66 Src homologous-collagen homologue (p66shc) inside cancer cells. Mortalin and SHetA2 conflictingly regulate the same molecules involved in mitochondria-mediated intrinsic apoptosis. The results validate the power of this protocol for revealing drug targets.