Multiple Functional Activities Research Articles

SARS-CoV-2 infection induces human milk antibodies capable of mediating multiple functional activities

SARS-CoV-2 infection induces human milk antibodies capable of mediating multiple functional activities

Wireless pressure insoles for measuring ground reaction forces and trajectories of the centre of pressure during functional activities

Wireless pressure insoles may enable the assessment of movement biomechanics in a real-world setting, and thus play an important role in the recommendation of clinical management, but they are not yet a gold standard due to the unknown accuracy and reliability with respect to different functional activities. Here, we compare novel wireless pressure insoles with force plates and examine the test–retest reliability of the insoles for measuring vertical ground reaction forces (vGRFs) and trajectories of the center of pressure (COP). In this observational study, healthy adults underwent two data collection sessions during one day. The Bland–Altman analysis was used to compare the outcomes measured with the two instruments during squats, jumps, and the sit-to-stand test. Test–retest reliability was assessed by the interclass correlation coefficient and the standard error of measurement for the outcomes during squats, jumps, walking, and stair ambulation. Trajectories of the COP in the anterior–posterior direction were comparable between the two systems during all activities. The insoles consistently measured shorter trajectories of the COP in the medial–lateral direction (except jumps) and lower vGRFs than the force plates. Test–retest reliability of the insoles was fair to high or excellent for all outcomes during all activities. In conclusion, the insoles provide reliable measures of vGRFs and trajectories of the COP during multiple functional activities in healthy adults. Although the insoles do not produce identical results to the force plate, the qualitative similarity and consistency between the two systems confirm the insoles can be used to measure these outcomes, based on the purpose and accuracy required.

Type 1 secretion system and effectors in Rickettsiales.

Obligate intracellular bacteria in the order Rickettsiales are transmitted by arthropod vectors and cause life-threatening infections in humans and animals. While both type 1 and type 4 secretion systems (T1SS and T4SS) have been identified in this group, the most extensive studies of Rickettsiales T1SS and associated effectors have been performed in Ehrlichia. These studies have uncovered important roles for the T1SS effectors in pathobiology and immunity. To evade innate immune responses and promote intracellular survival, Ehrlichia and other related obligate pathogens secrete multiple T1SS effectors which interact with a diverse network of host targets associated with essential cellular processes. T1SS effectors have multiple functional activities during infection including acting as nucleomodulins and ligand mimetics that activate evolutionarily conserved cellular signaling pathways. In Ehrlichia, an array of newly defined major immunoreactive proteins have been identified that are predicted as T1SS substrates and have conformation-dependent antibody epitopes. These findings highlight the underappreciated and largely uncharacterized roles of T1SS effector proteins in pathobiology and immunity. This review summarizes current knowledge regarding roles of T1SS effectors in Rickettsiales members during infection and explores newly identified immunoreactive proteins as potential T1SS substrates and targets of a protective host immune response.

Untargeted Lipidomics Revealed the Protective Effects of Cyanidin-3-O-glucoside on Bisphenol A-Induced Liver Lipid Metabolism Disorder in Rats.

Bisphenol A (BPA) is an estrogenic endocrine disruptor that induces metabolic disorders. Cyanidin-3-O-glucoside (C3G) has multiple functional activities and is the most abundant anthocyanin belonging to the flavonoid subgroup. This study aimed to investigate the protective effect of C3G on BPA-induced liver lipid metabolism disorder and explore its mechanism via lipidomics analysis. The results showed that C3G supplementation significantly ameliorated the serum levels of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total cholesterol, triacylglycerols (TG), and alanine and aspartate aminotransferase (ALT and AST). Furthermore, liver lipidomics indicated that C3G effectively facilitated the recovery of differential lipid metabolites, including TGs, phosphatidylethanolamines, phosphatidylcholines, lysophosphatidylcholines, phosphatidylinositol, cholesteryl esters, and phosphatidylserine, and reversed the levels of hepatic lipid synthesis-related genes. Our results suggest that C3G has an effective regulatory effect on BPA-induced disorders of lipid metabolism.

Characterization and efficient production of an α‐agarase from marine bacterium Catenovulum maritimum STB14

AbstractAgarases are enzymes that degrade agar and agarose to produce agar oligosaccharides with multiple functional activities. Compared with β‐agarases, the natural source of α‐agarases is limited, which severely restricts the industrial application of α‐agarases. Here, we cloned and heterologously expressed an α‐agarase belonging to glycoside hydrolase family 96 named Cm‐AGA from the marine bacterium Catenovulum maritimum STB14. The production conditions of recombinant Cm‐AGA were optimized as: taking Terrific Broth (TB) (pH 6.5) with 5 g/L of fructose and 24 g/L of yeast extract H07014 as the fermentation medium, after culturing at 37°C for 2 h, isopropyl‐β‐ d‐thiogalactoside was added with a final concentration of 0.01 mM to induce for 44 h. The obtained enzyme activity was 13.81 U/ml and was about 6.6 times the initial activity. The specific activity of recombinant Cm‐AGA was 206.1 U/mg, the optimum temperature and pH were 35°C and 8.0, respectively, and the enzyme activity could be activated by Mn2+ and Ca2+. The hydrolysis product results showed that Cm‐AGA is the first reported α‐agarase with agarobiose (A2) and agarotetraose (A4) as the dominant products, suggesting the great potential of Cm‐AGA in the efficient production of agaro‐oligosaccharides with a low degree of polymerization.

Abnormal Regional Spontaneous Neural Activity and Functional Connectivity in Unmedicated Patients with Narcolepsy Type 1: A Resting-State fMRI Study.

Previous Resting-state functional magnetic resonance imaging (fMRI) studies have mainly focused on cerebral functional alteration in processing different emotional stimuli in patients with narcolepsy type 1 (NT1), but were short of exploration of characteristic brain activity and its remote interaction patterns. This study aimed to investigate the spontaneous blood oxygen fluctuations at rest and to elucidate the neural mechanisms underlying neuropsychiatric behavior. A total of 18 unmedicated patients with NT1 and matched healthy individuals were recruited in a resting-state fMRI study. Magnetic resonance imaging (MRI) data were first analyzed using fractional low-frequency amplitude of low-frequency fluctuation (fALFF) to detect changes in local neural activity, and regions with group differences were taken as regions of interest (ROIs). Secondly, functional connectivity (FC) analysis was used to explore altered connectivity between ROIs and other areas. Lastly, the relationship between functional brain activity and neuropsychiatric behaviors was analyzed with correlation analysis. fALFF analysis revealed enhanced neural activity in bilateral fusiform gyrus (FFG), right precentral gyrus, and left postcentral gyrus (PoCG) in the NT1 group. The patients indicated reduced activity in the bilateral temporal pole middle temporal gyrus (TPOmid), left caudate nucleus (CAU), left parahippocampus, left precuneus (PCUN), right amygdala, and right anterior cingulate and paracingulate gyri. ESS score was negatively correlated with fALFF in the right FFG. The NT1 group revealed decreased connectivity between left TPOmid and right PoCG, the bilateral middle frontal gyrus, left superior frontal gyrus, medial, and right supramarginal gyrus. Epworth Sleepiness Scale (ESS) was negatively correlated with FC of the left TPOmid with left putamen (PUT) in NT1. Compared with healthy controls (HCs), enhanced FC of the left CAU with right FFG was positively associated with MSLT-SOREMPs in patients. Furthermore, increased FC of the left PCUN with right PoCG was positively correlated with SDS score. We found that multiple functional activities related to the processing of emotional regulation and sensory information processing were abnormal, and some were related to clinical characteristics. fALFF in the left postcentral or right precentral gyrus may be used as a biomarker of narcolepsy, whereas fALFF in the right fusiform and the FC strength of the left temporal pole middle temporal gyrus with the putamen may be clinical indicators to assess the drowsiness severity of narcolepsy.

Validity and reliability of accelerations and orientations measured using wearable sensors during functional activities

Wearable sensors may enable the assessment of movement in a real-world setting, but they are not yet a standard practice in the analysis of movement due to the unknown accuracy and reliability with respect to different functional activities. Here, we established the concurrent validity and test–retest reliability of accelerations and orientations measured using affordable novel sensors during squats, jumps, walking and stair ambulation. In this observational study, participants underwent three data collection sessions during one day. Accelerations and orientations from sacrum, thigh and shank were collected using these sensors and already validated gold-standard sensors as the criterion method. We assessed validity by comparing the similarity of signal waveforms with the Linear Fit Method and by comparing mean differences in range values with the Bland–Altman plots. Reliability was assessed by calculating interclass correlation coefficient and standard error of measurements of the range values. Concurrent validity was from fair to excellent in 91% of the cases for accelerations and in 84.4% for orientations. Test–retest reliability of accelerations was from fair to excellent in 97% of cases when the sensors were attached by a researcher, and in 84.4% of cases when the sensors were attached by participants. Test–retest reliability of orientations was from fair to excellent in 88.9% of cases when the sensors were attached by a researcher, and in 68.9% of cases when the sensors were attached by participants. In conclusion, the new affordable sensors provide accurate measures of accelerations and orientations during multiple functional activities in healthy adults. Reliability of the orientations may depend on the ability to replicate the same position of the sensor under test–retest conditions.

Unexpected Off-Target Activities for Recombinant C5a in Human Macrophages

The anaphylatoxin C5a is core effector of complement activation. C5a exerts potent proinflammatory and immunomodulatory actions through interacting with its C5a receptors, C5aR1 and C5aR2, modulating multiple signaling and functional activities of immune cells. Native C5a contains a large N-linked glycosylation site at Asn64, which accounts for up to 25% of its m.w. To date, the vast majority of published studies examining C5a are performed using Escherichia coli-generated recombinant C5a, which is readily available from numerous commercial suppliers, but lacks this glycosylation moiety. However, a plasma-purified "native" form of C5a is also commercially available. The different size and glycosylation of these two C5a versions could have functional implications. Therefore, the current study aimed to compare recombinant human C5a to purified plasma-derived human C5a in driving the signaling and functional activities of human primary macrophages. We found that both versions of C5a displayed similar potencies at triggering C5aR1- and C5aR2-mediated cell signaling, but elicited distinct functional responses in primary human monocyte-derived macrophages. Multiple commercial sources of recombinant C5a, but not the plasma-purified or a synthetic C5a version, induced human monocyte-derived macrophages to produce IL-6 and IL-10 in a C5a receptor-independent manner, which was driven through Syk and NF-κB signaling and apparently not due to endotoxin contamination. Our results, therefore, offer caution against the sole use of recombinant human C5a, particularly in functional/cytokine assays conducted in human primary immune cells, and suggest studies using recombinant human C5a should be paired with C5aR1 inhibitors or purified/synthetic human C5a to confirm relevant findings.

Construction and optimization of cordycepin-producing Saccharomyces cerevisiae

Cordycepin is the key active component of medicinal fungus Cordyceps militaris, and it shows multiple functional activities such as anti-tumor and anti-virus. Cordycepin was conventionally produced by liquid fermentation of C. militaris, but the long production cycle and the low productivity constrained its development and application. In this study, two key genes for cordycepin biosynthesis (ScCNS1 and ScCNS2) were introduced into Saccharomyces cerevisiae S288C, producing 67.32 mg/L cordycepin at 240 h. Analysis of gene expression profiles indicated that ZWF1, PRS4, ADE4, ScCNS1 and ScCNS2 which encode enzymes involved in pentose phosphate pathway, purine metabolism and cordycepin biosynthesis pathway, were significantly up-regulated in the late phage of fermentation. Optimization of fermentation medium determined that 50 g/L initial glucose followed by feeding, supplemented with 5 mmol/L Cu²⁺ and 1.0 g/L adenine were the best condition. Fed-batch fermentation using the engineered yeast in a 5 L stirred fermenter produced 137.27 mg/L cordycepin at 144 h, with a productivity up to 0.95 mg/(L·h) reached, which was 240% higher than that of the control.

Fucoxanthin@Polyvinylpyrrolidone Nanoparticles Promoted Oxidative Stress-Induced Cell Death in Caco-2 Human Colon Cancer Cells.

Fucoxanthin (FX), a natural carotenoid found in seaweed with multiple functional activities, is unstable with a poor water solubility that limits its utilization. This study aimed to improve FX’s stability and bioavailability via the nano-encapsulation of FX in polyvinylpyrrolidone (PVP)-coated FX@PVP nanoparticles (NPs). The FX@PVP NPs were evaluated in terms of their morphology, stability, encapsulation efficiency (EE), loading capacity (LC), and in vitro release to optimize the encapsulation parameters, and a 1:8 FX:PVP ratio was found to perform the best with the highest EE (85.50 ± 0.19%) and LC (10.68 ± 0.15%) and improved FX stability. In addition, the FX@PVP NPs were shown to effectively deliver FX into Caco-2 cancer cells, and the accumulation of FX in these cancer cells showed pro-oxidative activities to ameliorate H2O2-induced damage and cell death. The FX@PVP NPs could potentially become a new therapeutical approach for targeted cancer treatment.

A Preliminary Measurement and Understanding of Movement-Evoked Pain in African American Elder Adults

Osteoarthritis (OA) is a principal cause of disabling knee pain, and movement is a known exacerbator of pain in African Americans (AAs). Still, research has neglected to understand the relationship between pain with movement and its impact on function and mobility. Our previous study found significantly higher movement-evoked pain between AAs and White American (WAs). Therefore, this case-control observational study investigated inter-racial and intra-racial differences in movement-evoked pain in AAs and WAs (N= 28) who were 55-78 years-of-age (M= 65.75, SD= 6.23). We measured pain intensity (0-10) pre/ante/post multiple performance-based functional activities; we report preliminary results for 7-meter GAITRite® walk and Stair climbs. Pain intensity was higher before and after the 7-meter walk and stair climbs in AAs, although not significantly different than WAs. We will conduct additional statistical tests for the remaining functional activities to identify potential differences and ethnic-specific factors that distinguish movement-evoked pain and function by race.

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure and recurrence in acute myeloid leukemia (AML). Pleiotropic cytokines like interleukin (IL)-3 dimerize their receptors to signal multiple functional activities of AML cells, however, the mechanistic link between cytokine receptor dimerization and LSC fate regulation is unknown. Here, we show that the Class I heterodimeric IL3Rα/βc receptors (IL-3R) assemble as hexamers and dodecamers that segregate proliferation versus differentiation signals, respectively. Receptor stoichiometry varies across the individual cells that make up the AML hierarchy, with LSCs presenting with the highest IL3Rα/βc ratio thereby driving resultant hexamer-mediated stemness programs and poor patient survival. Our study establishes a new signaling mechanism of cytokine pleiotropy and the biased instruction of cell fate that directs the AML hierarchy; a signaling mechanism that may be generalizable to other normal and transformed cellular hierarchies.

Overexpression of CpSIZ1, a SIZ/PIAS-Type SUMO E3 Ligase from Wintersweet (Chimonanthus praecox), Delays Flowering, Accelerates Leaf Senescence and Enhances Cold Tolerance in Arabidopsis

Wintersweet (Chimonanthus praecox L.) is a traditional winter-flowering plant in China and a popular cut flower in winter. Its unique flowering characteristics under cold stress may involve the regulation of a large number of proteins. Protein post-translational modification is an important regulating type for the gene function. However, little is known about the post-translational modification in wintersweet. SUMOylation is an important post-translational modification in eukaryotes. Small ubiquitin-like modifier (SUMO) E3 ligases perform multiple functional regulatory activities in plants via SUMOylation. Here, we cloned and identified a SIZ/PIAS-type SUMO E3 ligase, CpSIZ1, from wintersweet. CpSIZ1 shared high similarity with other SIZ1 proteins. Quantitative real-time PCR (qRT-PCR) indicated that CpSIZ1 was expressed in all tissues tested, with the highest expression in flower wither period of stage 6, and followed by mature leaves except for different flower development stages. The ectopic expression of CpSIZ1 in Arabidopsis, including the CpSIZ1 overexpression in siz1-2 mutant (HB line) and CpSIZ1 overexpression in WT (OE line), not only promoted vegetative growth, delayed flowering and accelerated leaf senescence, but also improve the cold tolerance in Arabidopsis. Therefore, our studies have enrich the understanding of function of SIZ1 gene in woody plant, and provide a good foundation for further research on the post-translational modification regulation mechanism in this winter-flowering plant.

Abstract A89: Identification of novel costimulatory pathways in T cells with functional assays on the Membrane Proteome Array (MPA)

Abstract To identify membrane proteins involved in cytotoxic T-cell (CTL) stimulation, we screened our proprietary Membrane Proteome Array (MPA) for activation of CTLs. The MPA comprises 5,300 different human membrane proteins, each overexpressed in live HEK-293 cells. CTLs were layered on the MPA, incubated overnight, stained for an activation marker, and quantified for expression by high-throughput flow cytometry. We identified 38 putative membrane proteins involved in CTL activation. Eight of these “hits” are known activators of CTLs, such as CD86. Other hits include membrane proteins that are involved in multiple functional activities. Here, we describe the functional validation of two of these previously unreported stimulatory proteins. For Hit A, we converted the extracellular domain to an Fc-fusion protein and used the construct to probe the MPA for binding partners. We identified two proteins that interact with Hit A: one protein found on neurons and one protein found on T cells. Hit B was a commercially available molecular agonist. Using the agonist in our assay, we were able to modulate the extent to which this target stimulated CTLs. In addition to identifying factors of CTL activation, we are currently using the MPA to identify novel viral receptors and therapeutic targets for neurodegenerative diseases, as well as deorphaning and testing the specificity of antibodies. Citation Format: Jonathan T. Sullivan, Duncan Huston-Paterson, Charles Azuelos, Benjamin J. Doranz. Identification of novel costimulatory pathways in T cells with functional assays on the Membrane Proteome Array (MPA) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A89.

Biomechanical analysis of the timed up-and-go (TUG) test in children with and without Down syndrome

BackgroundThe timed up-and-go (TUG) test consists of multiple functional activities of daily living performed in a sequence, with the goal to complete the test as quickly as possible. Considering children with Down syndrome (DS) have been shown to take longer to complete the TUG test, it is imperative to identify which tasks are problematic for this population in order to individualize physical interventions. Research questionIs the biomechanical pattern of each functional task during the TUG test different between children with DS and typically developing (TD) children? MethodsThirteen children with DS and thirteen TD children aged 5–11 years old completed the TUG test. Kinematic data was captured using a Vicon motion capture system. We visually coded the TUG test into five phases: sit-to-stand, walk-out, turn-around, walk-in, and stand-to-sit. We focused on the center-of-mass (COM) movement in the sit-to-stand phase, spatiotemporal parameters in the walk-out phase, and intersegmental coordination in the turn-around phase. Results and significanceChildren with DS took longer to complete the entire test, as well as each of the five phases. During the sit-to-stand phase, children with DS produced smaller peak vertical COM velocity, medial-lateral COM excursion, and peak knee and hip extension velocity compared to TD peers. Children with DS walked at a slower velocity during the walk-out phase. Both groups demonstrated a similar intersegmental coordination pattern between the head, thorax, and pelvis during the turn-around phase although children with DS had slower average and peak angular velocity at the head, thorax, and pelvis. Our results suggest that children with DS were less able to anticipate transitioning between motor tasks and took longer to initiate motor tasks. Our TUG analysis provides the detailed insights to help evaluate individual motor tasks as well as the transition from one task to another for clinical populations.

Acetylation is the most abundant actin modification in Entamoeba histolytica and modifications of actin's amino-terminal domain change cytoskeleton activities.

Actin is one of the most conserved, abundant, and ubiquitous proteins in all eukaryotes characterised to date. Posttranslation modifications of actin modify the organisation of the actin-rich cytoskeleton. In particular, chemical modifications of actin's amino-terminal region determine how filamentous actin is organised into scaffolds. After assuming that protein modifications account for the multiple functional activities exerted by the single actin in Entamoeba histolytica, we profiled posttranslational modifications of this protein. Acetylation (on 21 different amino acids) was the most abundant modification, followed by phosphorylation. Furthermore, the glycine residue at Position 2 in E. histolytica's actin (Gly2, not found in most other eukaryotic actins) was found to be acetylated. The impact of Gly2 on the amoeba's life cycle and pathogenicity was then assessed in mutagenesis experiments. We found that Gly2 was necessary for cell morphology and division, parasite-host cell adhesion, and host invasion in an in vitro model of amoebic human infection.

Contemporary understanding of the secretory granules in human eosinophils.

Eosinophil secretory (specific) granules have a unique morphology and are both a morphologic hallmark of eosinophils and fundamental to eosinophil-mediated responses. Eosinophil mediators with multiple functional activities are presynthesized and stored within these granules, poised for very rapid, stimulus-induced secretion. The structural organization and changes of eosinophil specific granules are revealing in demonstrating the complex and diverse secretory activities of this cell. Here, we review our current knowledge on the architecture, composition, and function of eosinophil specific granules as highly elaborated organelles able to produce vesiculotubular carriers and to interplay with the intracellular vesicular trafficking. We reconsider prior identifications of eosinophil cytoplasmic granules, including "primary," "secondary," "microgranules," and "small granules"; and consonant with advances, we provide a contemporary recognition that human eosinophils contain a single population of specific granules and their developmental precursors and derived secretory vesicles.

Neutralizing Antibodies Against a Specific Human Immunodeficiency Virus gp41 Epitope are Associated With Long-term Non-progressor Status

Antibodies (Abs) play a central role in human immunodeficiency virus (HIV) protection due to their multiple functional inhibitory activities. W614A-3S Abs recognize a specific form of a highly conserved motif of the gp41 envelope protein and can elicit viral neutralization to protect CD4+ T cells. Here, we describe in detail the neutralizing profile of W614A-3S Abs in untreated long-term non-progressor (LTNP) HIV-infected patients. W614A-3S Abs were detected in 23.5% (16/68) of untreated LTNP patients compared with <5% (5/104) of HIV-1 progressor patients. The W614A-3S Abs had efficient neutralizing activity that inhibited transmitted founder primary viruses and exhibited Fc-mediated inhibitory functions at low concentrations in primary monocyte-derived macrophages. The neutralizing capacity of W614A-3S Abs was inversely correlated with viral load (r=−0.9013; p<0.0001), viral DNA (r=−0.7696; p=0.0005) and was associated the preservation of high CD4+ T-cell counts and T-cell responses. This study demonstrates that W614A-3S neutralizing Abs may confer a crucial advantage to LTNP patients. These results provide insights for both pathophysiological research and the development of vaccine strategies.

Convergent validity of the Timed Up and Go Test and Ten-metre Timed Walk Test in pregnant women with pelvic girdle pain

Pregnant women with pelvic girdle pain (PGP) often experience functional difficulties, in particular walking difficulties. Currently, however, there is a lack of validated performance-orientated outcome measures available for use in this population. The Timed Up and Go (TUG) test and Ten-metre Timed Walk Test (10 mTWT) are two short-distance walking tests that have demonstrated reliability in pregnant women with PGP, but as yet have no established validity. The aim of the present study was to evaluate the convergent validity of the TUG and 10 mTWT by comparing performances on these two walking tests with scores achieved on the Active Straight Leg Raise (ASLR) test and the Pelvic Girdle Questionnaire (PGQ). Eighteen pregnant women with PGP aged 31.4 years (SD = 2.7) and 28.9 weeks pregnant (SD = 7.3) were included. Spearman rank correlation coefficient (rs) was used to determine convergent validity. Strong correlations were found between the TUG and ASLR (rs = 0.73, p = 0.001), and the 10 mTWT and ASLR (rs = −0.65, p = 0.003). Relationships between the TUG and PGQ were moderate (rs = 0.41 to 0.52) and between the 10 mTWT and PGQ low to moderate (rs = −0.25 to −0.56). The strong relationships between the walking tests and the ASLR may suggest these tests all assess the same construct. The weaker relationships found between the walking tests and the PGQ may be related to the self-report and multiple functional activities nature of the questionnaire. This study found both the TUG and 10 mTWT to be valid weight-bearing physical performance measures, although more research is warranted due to the small study sample.

Automated quantitative multiplex immunofluorescence in situ imaging identifies phospho-S6 and phospho-PRAS40 as predictive protein biomarkers for prostate cancer lethality.

BackgroundWe have witnessed significant progress in gene-based approaches to cancer prognostication, promising early intervention for high-risk patients and avoidance of overtreatment for low-risk patients. However, there has been less advancement in protein-based approaches, even though perturbed protein levels and post-translational modifications are more directly linked with phenotype. Most current, gene expression-based platforms require tissue lysis resulting in loss of structural and molecular information, and hence are blind to tumor heterogeneity and morphological features.ResultsHere we report an automated, integrated multiplex immunofluorescence in situ imaging approach that quantitatively measures protein biomarker levels and activity states in defined intact tissue regions where the biomarkers of interest exert their phenotype. Using this approach, we confirm that four previously reported prognostic markers, PTEN, SMAD4, CCND1 and SPP1, can predict lethal outcome of human prostate cancer. Furthermore, we show that two PI3K pathway-regulated protein activities, pS6 (RPS6-phosphoserines 235/236) and pPRAS40 (AKT1S1-phosphothreonine 246), correlate with prostate cancer lethal outcome as well (individual marker hazard ratios of 2.04 and 2.03, respectively). Finally, we incorporate these 2 markers into a novel 5-marker protein signature, SMAD4, CCND1, SPP1, pS6, and pPRAS40, which is highly predictive for prostate cancer-specific death. The ability to substitute PTEN with phospho-markers demonstrates the potential of quantitative protein activity state measurements on intact tissue.ConclusionsIn summary, our approach can reproducibly and simultaneously quantify and assess multiple protein levels and functional activities on intact tissue specimens. We believe it is broadly applicable to not only cancer but other diseases, and propose that it should be well suited for prognostication at early stages of pathogenesis where key signaling protein levels and activities are perturbed.