Small-Scale Cultivation and Transfection of ExpiCHO and HEK293E Cells in Single-Use Orbitally Shaken Bioreactors.

Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the two most important mammalian hosts for the production of recombinant proteins. In this chapter, the suspension cultivation and transfection of these cells in small-scale, single-use orbitally shaken bioreactors, TubeSpin™ bioreactor 50 [orbitally shaken reactor 50 (OSR50)], and TubeSpin™ bioreactor 600 [orbitally shaken reactor 600 (OSR600)] are described. These are conical centrifuge tubes with nominal volumes of 50 mL and 600 mL, respectively, that have been redesigned with a ventilated cap for the cultivation of animal cells in suspension at working volumes up to 20 mL and 400 mL, respectively.

Examining the validity of the Delis-Kaplan Executive Function System (D-KEFS) in traumatic brain injury.

This study examines the validity of the Delis-Kaplan Executive Function System (D-KEFS) in a traumatic brain injury (TBI) population compared to participants with orthopaedic injuries and normative controls. The utility of the D-KEFS was examined using a between groups design. One hundred patients with mild uncomplicated to severe TBI were recruited from a consecutive cohort of patients admitted as inpatients to a UK Major Trauma Centre and compared to 823 participants from the D-KEFS normative sample and 26 participants with orthopaedic injuries. Data were filtered for performance validity. Sample discrimination was calculated from D-KEFS subtest scores and derived index scores. Sensitivity to TBI severity was established. The TBI participants performed significantly lower on the D-KEFS Trail Making Test, Colour Word Interference, Colour Word Switching, Letter Fluency and Verbal Fluency Category Switching Total Words Correct. The D-KEFS index scores discriminated between TBI, orthopaedic and normative participants with large and moderate effect sizes, respectively. The D-KEFS demonstrated a dose-response relationship with TBI severity. These effects were robust to differences in premorbid intellectual functioning; however, D-KEFS performance was sensitive to performance on tests of mental processing speed. The use of a D-KEFS index score provides a robust and reliable discrimination of TBI patients from healthy control participants. This discrimination is not accounted for by premorbid intellect or the non-specific effects of trauma. The clinical and conceptual implications of these findings are considered.

A meta-analytic review examining the validity of executive functioning tests to predict functional outcomes in individuals with a traumatic brain injury.

OBJECTIVES: Deficits in executive functioning are a common consequence of Traumatic Brain Injury (TBI) and the severity of TBI is known to predict functional outcomes. In this review, the authors examine the ability of three commonly used tests of executive functioning [The Trail Making Test (TMT-B), The Wisconsin Card Sorting Test (WCST), and Verbal Fluency (VF)] to predict domains of function. METHODS: Seven hundred and twenty articles were identified and twenty-four met inclusion criteria (original articles published in English examining an adult TBI population). Data were subject to a study quality analysis and then meta-analyzed to assess whether tests of executive functioning (TMT-B, WCST, and VF) can predict functional, employment, and driving outcomes following a TBI. RESULTS: The TMT-B (r = 0.29; 95% CI 0.17-0.41) and the WCST (r = 0.20; 95% CI 0.02-0.37) were significantly associated with functional outcomes. The TMT-B was also associated with a person's ability to return to driving (r = 0.3890; 95% CI 0.2678-0.5103). No test of executive functioning was associated with employment outcomes following a TBI. CONCLUSION: These findings are important to guide rehabilitation strategies and future planning. This review has also highlighted the scarcity of research on specific outcomes.

Cognitive Outcome After Complicated Mild Traumatic Brain Injury: A Literature Review and Meta-Analysis.

Cognitive outcome for mild traumatic brain injury (mTBI) with positive brain imaging (complicated mTBI) was compared with that for mTBI with normal imaging (uncomplicated mTBI) and with moderate to severe TBI, using meta-analysis. Twenty-three studies utilizing objective neurocognitive tests were included in the analysis. At less than 3 months post-injury, complicated mTBI was associated with poorer cognitive outcomes than uncomplicated mTBI, but deficits were not comparable to those with moderate-severe TBI. After 3 months post-injury, a similar pattern was detected. Beyond 3 months, deficits in complicated mTBI relative to those with uncomplicated mTBI were present in processing speed, memory, executive function, and language, although the latter may be the result of reduced semantic fluency. The effect size of deficits in these domains was more marked in moderate-severe TBI. The available data support the use of complicated mTBI as a distinct classification in the prediction of cognitive outcome. The extent of cognitive deficit in complicated mTBI was small and unlikely to cause significant disability. However, patients with complicated mTBI constitute a broad category encompassing individuals who may differ markedly in the nature and extent of intracranial imaging abnormality, and further studies are warranted. Limitations of the available studies include small, selected samples; variations in TBI severity classification; absence of validity ("effort") testing; differing imaging methodology; and lack of long-term follow-up.

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

An evaluation of the convergent validity of a face-to-face and virtual neuropsychological assessment counter balanced.

The COVID-19 pandemic has highlighted the need for further research evaluating the validity of conducting a battery of neuropsychological assessments virtually compared with face-to-face administration. Previous research has suggested that some neuropsychological assessments yield valid results when administered virtually, however, much of the previous research focused on older adults. To determine the validity of virtually administered neuropsychological tests, 28 healthy participants were assessed using a within-subjects, counter-balanced design. Participants completed a neuropsychological assessment battery covering tests of general intellectual functioning, memory and attention, executive functioning, language and information processing speed, as well as effort. There was no significant difference between face-to-face administration of the neuropsychological battery compared with virtual administration for the majority of the tests used. However, there were significant differences in the Colour Naming Task, with participants making fewer errors on the colour naming task and inhibition/switching task when administered virtually compared with face-to-face administration. There was also a significant age cohort effect in the inhibition/switching task. There was also a trending significant difference in mode of administration for the Verbal Fluency Task. Virtually administered neuropsychological assessments largely provide a valid alternative to face-to-face assessments; however, consideration must be given to test selection as well as the population of participants that are being assessed. Other important considerations must focus on preserving the security and integrity of test materials, as well as administration in a medico-legal setting. Future research should focus on validating assessments with specific patient populations and developing a neuropsychological assessment battery using information technology.

Validation of the Neuropsychological Assessment Battery Screening Module (NAB-SM) in patients with traumatic brain injury.

This study cross-validates the screening module of the Neuropsychological Assessment Battery (NAB-SM) with a battery of well validated neuropsychological tests (Convergent Validity Test Battery: CVTB) in a Traumatic Brain Injury (TBI) population. Forty-four participants with "mild-complicated" to "severe" TBI were recruited from a cohort of patients attending an outpatient clinic at a UK major trauma center. The NAB-SM Total Index score and an abbreviated short-form, from which a TBI Index was derived, both showed good classification accuracy in predicting impairment as measured by the CVTB mean score. These indices also accurately identified impairment as defined by the base rate of low scores across individual CVTB indices measuring mental processing speed, working memory, memory and executive functioning. The NAB-SM and its derived TBI index therefore have significant utility as a cognitive screening tool for use in either inpatient (acute) or outpatient TBI populations.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and ß-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small ß-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The ß-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the ß-annulus peptide for vaccine design.

Changes in SARS-CoV-2 Spike versus Nucleoprotein Antibody Responses Impact the Estimates of Infections in Population-Based Seroprevalence Studies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses to the spike (S) protein monomer, S protein native trimeric form, or the nucleocapsid (N) proteins were evaluated in cohorts of individuals with acute infection (n = 93) and in individuals enrolled in a postinfection seroprevalence population study (n = 578) in Switzerland. Commercial assays specific for the S1 monomer, for the N protein, or within a newly developed Luminex assay using the S protein trimer were found to be equally sensitive in antibody detection in the acute-infection-phase samples. Interestingly, compared to anti-S antibody responses, those against the N protein appear to wane in the postinfection cohort. Seroprevalence in a "positive patient contacts" group (n = 177) was underestimated by N protein assays by 10.9 to 32.2%, while the "randomly selected" general population group (n = 311) was reduced by up to 45% relative to the S protein assays. The overall reduction in seroprevalence targeting only anti-N antibodies for the total cohort ranged from 9.4 to 31%. Of note, the use of the S protein in its native trimer form was significantly more sensitive compared to monomeric S proteins. These results indicate that the assessment of anti-S IgG antibody responses against the native trimeric S protein should be implemented to estimate SARS-CoV-2 infections in population-based seroprevalence studies.IMPORTANCE In the present study, we have determined SARS-CoV-2-specific antibody responses in sera of acute and postinfection phase subjects. Our results indicate that antibody responses against viral S and N proteins were equally sensitive in the acute phase of infection, but that responses against N appear to wane in the postinfection phase where those against the S protein persist over time. The most sensitive serological assay in both acute and postinfection phases used the native S protein trimer as the binding antigen, which has significantly greater conformational epitopes for antibody binding compared to the S1 monomer protein used in other assays. We believe these results are extremely important in order to generate correct estimates of SARS-CoV-2 infections in the general population. Furthermore, the assessment of antibody responses against the trimeric S protein will be critical to evaluate the durability of the antibody response and for the characterization of a vaccine-induced antibody response.

Tomographic Task-Related Functional Near-Infrared Spectroscopy in Acute Sport-Related Concussion: An Observational Case Study.

Making decisions regarding return-to-play after sport-related concussion (SRC) based on resolution of symptoms alone can expose contact-sport athletes to further injury before their recovery is complete. Task-related functional near-infrared spectroscopy (fNIRS) could be used to scan for abnormalities in the brain activation patterns of SRC athletes and help clinicians to manage their return-to-play. This study aims to show a proof of concept of mapping brain activation, using tomographic task-related fNIRS, as part of the clinical assessment of acute SRC patients. A high-density frequency-domain optical device was used to scan 2 SRC patients, within 72 h from injury, during the execution of 3 neurocognitive tests used in clinical practice. The optical data were resolved into a tomographic reconstruction of the brain functional activation pattern, using diffuse optical tomography. Moreover, brain activity was inferred using single-subject statistical analyses. The advantages and limitations of the introduction of this optical technique into the clinical assessment of acute SRC patients are discussed.

Analysis of volumetric mass transfer coefficient (k L a) in small- (250 mL) to large-scale (2500 L) orbitally shaken bioreactors.

In this study, the combination of dimensional analysis (DA) and analysis of variance (ANOVA) was used to predict the volumetric mass transfer coefficient (k L a) values under different operating conditions for orbitally shaken bioreactors (OSRs) with different filling volumes. It was found that Reynolds number and the interaction between Froude number and geometric number have the largest impact on k L a with impact indexes of 7.41 and 7.50, respectively. Moreover, the volume number has the largest negative impact on k L a, with an impact index of - 5.34. Thus, an effective way to increase the oxygen supply is by increasing the shaking speed and shaking diameter or decreasing the vessel diameter. However, cell cultivation with a higher filling volume will have an increased risk of oxygen scarcity. Therefore, with the help of the k L a prediction model, a suitable operating condition can be determined effectively and easily.

Large-Scale Production of Recombinant Noggin and R-Spondin1 Proteins Required for the Maintenance of Stem Cells in Intestinal Organoid Cultures.

The presence of the proteins mouse R-Spondin1 (mRSpo1) and mouse Noggin (mNoggin) in a 3D-organoid culture allows for the maintenance of intestinal stem cells. Here, we describe a transient gene expression method for the production of these proteins from human embryo kidney 293 (HEK293) cells cultivated in suspension using orbitally shaken bioreactors. Plasmid DNA was delivered into cells using the cationic polymer polyethylenimine (PEI). The 7-day production cultures were performed in the presence of valproic acid (VPA), an enhancer of recombinant gene expression. Both proteins were secreted from the transfected cells. mRSpo1 was produced as a secreted Fc fusion protein (mRSpo1-Fc) and purified by protein A-based affinity chromatography. mNoggin was produced as a secreted histidine-tagged protein (mNoggin-His) and purified by immobilized metal affinity chromatography (IMAC). This transient transfection system supports a high production efficiency.

Direct, Competitive Comparison of Linear, Monocyclic, and Bicyclic Libraries Using mRNA Display.

Peptide macrocyclization is typically associated with the development of higher affinity and more protease stable protein ligands, and, as such, is an important tool in peptide drug discovery. Yet, within the context of a diverse library, does cyclization give inherent advantages over linear peptides? Here, we used mRNA display to create a peptide library of diverse ring sizes and topologies (monocyclic, bicyclic, and linear). Several rounds of in vitro selection against streptavidin were performed and the winning peptide sequences were analyzed for their binding affinities and overall topologies. The effect of adding a protease challenge on the enrichment of various peptides was also investigated. Taken together, the selection output yields insights about the relative abundance of binders of various topologies within a structurally diverse library.

BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing.

Despite its widespread use, RNA-seq is still too laborious and expensive to replace RT-qPCR as the default gene expression analysis method. We present a novel approach, BRB-seq, which uses early multiplexing to produce 3' cDNA libraries for dozens of samples, requiring just 2 hours of hands-on time. BRB-seq has a comparable performance to the standard TruSeq approach while showing greater tolerance for lower RNA quality and being up to 25 times cheaper. We anticipate that BRB-seq will transform basic laboratory practice given its capacity to generate genome-wide transcriptomic data at a similar cost as profiling four genes using RT-qPCR.

Cognitive behavioural therapy plus standard care versus standard care for people with schizophrenia.

BACKGROUND: Cognitive behavioural therapy (CBT) is a psychosocial treatment that aims to re-mediate distressing emotional experiences or dysfunctional behaviour by changing the way in which a person interprets and evaluates the experience or cognates on its consequence and meaning. This approach helps to link the person's feelings and patterns of thinking which underpin distress. CBT is now recommended by the National Institute for Health and Care Excellence (NICE) as an add-on treatment for people with a diagnosis of schizophrenia. This review is also part of a family of Cochrane CBT reviews for people with schizophrenia. OBJECTIVES: To assess the effects of cognitive behavioural therapy added to standard care compared with standard care alone for people with schizophrenia. SEARCH METHODS: We searched the Cochrane Schizophrenia Group's Trials Register (up to March 6, 2017). This register is compiled by systematic searches of major resources (including AMED, BIOSIS CINAHL, Embase, MEDLINE, PsycINFO, PubMed, and registries of clinical trials) and their monthly updates, handsearches, grey literature, and conference proceedings, with no language, date, document type, or publication status limitations for inclusion of records into the register. SELECTION CRITERIA: We selected all randomised controlled clinical trials (RCTs) involving people diagnosed with schizophrenia or related disorders, which compared adding CBT to standard care with standard care given alone. Outcomes of interest included relapse, rehospitalisation, mental state, adverse events, social functioning, quality of life, and satisfaction with treatment.We included studies fulfilling the predefined inclusion criteria and reporting useable data. DATA COLLECTION AND ANALYSIS: We complied with the Cochrane recommended standard of conduct for data screening and collection. Where possible, we calculated relative risk (RR) and its 95% confidence interval (CI) for binary data and mean difference (MD) and its 95% confidence interval for continuous data. We assessed risk of bias for included studies and created a 'Summary of findings' table using GRADE. MAIN RESULTS: This review now includes 60 trials with 5,992 participants, all comparing CBT added to standard care with standard care alone. Results for the main outcomes of interest (all long term) showed no clear difference between CBT and standard care for relapse (RR 0.78, 95% CI 0.61 to 1.00; participants = 1538; studies = 13, low-quality evidence). Two trials reported global state improvement. More participants in the CBT groups showed clinically important improvement in global state (RR 0.57, 95% CI 0.39 to 0.84; participants = 82; studies = 2 , very low-quality evidence). Five trials reported mental state improvement. No differences in mental state improvement were observed (RR 0.81, 95% CI 0.65 to 1.02; participants = 501; studies = 5, very low-quality evidence). In terms of safety, adding CBT to standard care may reduce the risk of having an adverse event (RR 0.44, 95% CI 0.27 to 0.72; participants = 146; studies = 2, very low-quality evidence) but appears to have no effect on long-term social functioning (MD 0.56, 95% CI -2.64 to 3.76; participants = 295; studies = 2, very low-quality evidence, nor on long-term quality of life (MD -3.60, 95% CI -11.32 to 4.12; participants = 71; study = 1, very low-quality evidence). It also has no effect on long-term satisfaction with treatment (measured as 'leaving the study early') (RR 0.93, 95% CI 0.77 to 1.12; participants = 1945; studies = 19, moderate-quality evidence). AUTHORS' CONCLUSIONS: Relative to standard care alone, adding CBT to standard care appears to have no effect on long-term risk of relapse. A very small proportion of the available evidence indicated CBT plus standard care may improve long term global state and may reduce the risk of adverse events. Whether adding CBT to standard care leads to clinically important improvement in patients' long-term mental state, quality of life, and social function remains unclear. Satisfaction with care (measured as number of people leaving the study early) was no higher for participants receiving CBT compared to participants receiving standard care. It should be noted that although much research has been carried out in this area, the quality of evidence available is poor - mostly low or very low quality and we still cannot make firm conclusions until more high quality data are available.

Cognitive behavioural therapy plus standard care versus standard care plus other psychosocial treatments for people with schizophrenia.

BACKGROUND: Cognitive behavioural therapy (CBT) is a psychosocial treatment that aims to help individuals re-evaluate their appraisals of their experiences that can affect their level of distress and problematic behaviour. CBT is now recommended by the National Institute for Health and Care Excellence (NICE) as an add-on treatment for people with a diagnosis of schizophrenia. Other psychosocial therapies that are often less expensive are also available as an add-on treatment for people with schizophrenia. This review is also part of a family of Cochrane Reviews on CBT for people with schizophrenia. OBJECTIVES: To assess the effects of CBT compared with other psychosocial therapies as add-on treatments for people with schizophrenia. SEARCH METHODS: We searched the Cochrane Schizophrenia Group's Study Based Register of Trials (latest 6 March, 2017). This register is compiled by systematic searches of major resources (including AMED, BIOSIS CINAHL, Embase, MEDLINE, PsycINFO, PubMed, and registries of clinical trials) and their monthly updates, handsearches, grey literature, and conference proceedings, with no language, date, document type, or publication status limitations for inclusion of records into the register. SELECTION CRITERIA: We selected randomised controlled trials (RCTs) involving people with schizophrenia who were randomly allocated to receive, in addition to their standard care, either CBT or any other psychosocial therapy. Outcomes of interest included relapse, global state, mental state, adverse events, social functioning, quality of life and satisfaction with treatment. We included trials meeting our inclusion criteria and reporting useable data. DATA COLLECTION AND ANALYSIS: We reliably screened references and selected trials. Review authors, working independently, assessed trials for methodological quality and extracted data from included studies. We analysed dichotomous data on an intention-to-treat basis and continuous data with 60% completion rate. Where possible, for binary data we calculated risk ratio (RR), for continuous data we calculated mean difference (MD), all with 95% confidence intervals (CIs). We used a fixed-effect model for analyses unless there was unexplained high heterogeneity. We assessed risk of bias for the included studies and used the GRADE approach to produce a 'Summary of findings' table for our main outcomes of interest. MAIN RESULTS: The review now includes 36 trials with 3542 participants, comparing CBT with a range of other psychosocial therapies that we classified as either active (A) (n = 14) or non active (NA) (n = 14). Trials were often small and at high or unclear risk of bias. When CBT was compared with other psychosocial therapies, no difference in long-term relapse was observed (RR 1.05, 95% CI 0.85 to 1.29; participants = 375; studies = 5, low-quality evidence). Clinically important change in global state data were not available but data for rehospitalisation were reported. Results showed no clear difference in long term rehospitalisation (RR 0.96, 95% CI 0.82 to 1.14; participants = 943; studies = 8, low-quality evidence) nor in long term mental state (RR 0.82, 95% CI 0.67 to 1.01; participants = 249; studies = 4, low-quality evidence). No long-term differences were observed for death (RR 1.57, 95% CI 0.62 to 3.98; participants = 627; studies = 6, low-quality evidence). Only average endpoint scale scores were available for social functioning and quality of life. Social functioning scores were similar between groups (long term Social Functioning Scale (SFS): MD 8.80, 95% CI -4.07 to 21.67; participants = 65; studies = 1, very low-quality evidence), and quality of life scores were also similar (medium term Modular System for Quality of Life (MSQOL): MD -4.50, 95% CI -15.66 to 6.66; participants = 64; studies = 1, very low-quality evidence). There was a modest but clear difference favouring CBT for satisfaction with treatment - measured as leaving the study early (RR 0.86, 95% CI 0.75 to 0.99; participants = 2392; studies = 26, low quality evidence). AUTHORS' CONCLUSIONS: Evidence based on data from randomised controlled trials indicates there is no clear and convincing advantage for cognitive behavioural therapy over other - and sometimes much less sophisticated and expensive - psychosocial therapies for people with schizophrenia. It should be noted that although much research has been carried out in this area, the quality of evidence available is mostly low or of very low quality. Good quality research is needed before firm conclusions can be made.

CHO and HEK293 Cultivation and Transfection in Single-Use Orbitally Shaken Bioreactors.

Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the two most popular mammalian hosts for the production of recombinant proteins. In this chapter the suspension cultivation and transfection of these cells in small-scale disposable bioreactors is described. The TubeSpin bioreactor 50 and TubeSpin bioreactor 600 are designed for the cultivation of suspension cells by orbital shaking and have maximum working volumes of about 15 mL and 400 mL, respectively.

Improved process conditions for increasing expression of MHC class II protein from a stable Drosophila S2 cell line.

OBJECTIVES: To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. RESULTS: When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR12xHis) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested. CONCLUSIONS: Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.

Expression proteomics study to determine metallodrug targets and optimal drug combinations.

The emerging technique termed functional identification of target by expression proteomics (FITExP) has been shown to identify the key protein targets of anti-cancer drugs. Here, we use this approach to elucidate the proteins involved in the mechanism of action of two ruthenium(II)-based anti-cancer compounds, RAPTA-T and RAPTA-EA in breast cancer cells, revealing significant differences in the proteins upregulated. RAPTA-T causes upregulation of multiple proteins suggesting a broad mechanism of action involving suppression of both metastasis and tumorigenicity. RAPTA-EA bearing a GST inhibiting ethacrynic acid moiety, causes upregulation of mainly oxidative stress related proteins. The approach used in this work could be applied to the prediction of effective drug combinations to test in cancer chemotherapy clinical trials.