A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles.

We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.

Proteome census upon nutrient stress reveals Golgiphagy membrane receptors.

During nutrient stress, macroautophagy degrades cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodelling the proteome1,2. Although the machinery responsible for initiation of macroautophagy has been well characterized3,4, our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms, is limited. Here we use orthogonal proteomic strategies to provide a spatial proteome census of autophagic cargo during nutrient stress in mammalian cells. We find that macroautophagy has selectivity for recycling membrane-bound organelles (principally Golgi and endoplasmic reticulum). Through autophagic cargo prioritization, we identify a complex of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF3 and YIPF4 interact with ATG8 proteins through LIR motifs and are mobilized into autophagosomes that traffic to lysosomes in a process that requires the canonical autophagic machinery. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of a specific cohort of Golgi membrane proteins during nutrient stress. Moreover, YIPF3 and YIPF4 play an analogous role in Golgi remodelling during programmed conversion of stem cells to the neuronal lineage in vitro. Collectively, the findings of this study reveal prioritization of membrane protein cargo during nutrient-stress-dependent proteome remodelling and identify a Golgi remodelling pathway that requires membrane-embedded receptors.

PARK15/FBXO7 is dispensable for PINK1/Parkin mitophagy in iNeurons and HeLa cell systems.

The protein kinase PINK1 and ubiquitin ligase Parkin promote removal of damaged mitochondria via a feed-forward mechanism involving ubiquitin (Ub) phosphorylation (pUb), Parkin activation, and ubiquitylation of mitochondrial outer membrane proteins to support the recruitment of mitophagy receptors. The ubiquitin ligase substrate receptor FBXO7/PARK15 is mutated in an early-onset parkinsonian-pyramidal syndrome. Previous studies have proposed a role for FBXO7 in promoting Parkin-dependent mitophagy. Here, we systematically examine the involvement of FBXO7 in depolarization and mt UPR-dependent mitophagy in the well-established HeLa and induced-neurons cell systems. We find that FBXO7-/- cells have no demonstrable defect in: (i) kinetics of pUb accumulation, (ii) pUb puncta on mitochondria by super-resolution imaging, (iii) recruitment of Parkin and autophagy machinery to damaged mitochondria, (iv) mitophagic flux, and (v) mitochondrial clearance as quantified by global proteomics. Moreover, global proteomics of neurogenesis in the absence of FBXO7 reveals no obvious alterations in mitochondria or other organelles. These results argue against a general role for FBXO7 in Parkin-dependent mitophagy and point to the need for additional studies to define how FBXO7 mutations promote parkinsonian-pyramidal syndrome.

Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria.

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

Aquaporin-mediated dysregulation of cell migration in disease states.

Dysregulated cell migration and invasion are hallmarks of many disease states. This dysregulated migratory behavior is influenced by the changes in expression of aquaporins (AQPs) that occur during pathogenesis, including conditions such as cancer, endometriosis, and arthritis. The ubiquitous function of AQPs in migration of diseased cells makes them a crucial target for potential therapeutics; this possibility has led to extensive research into the specific mechanisms underlying AQP-mediated diseased cell migration. The functions of AQPs depend on a diverse set of variables including cell type, AQP isoform, disease state, cell microenvironments, and even the subcellular localization of AQPs. To consolidate the considerable work that has been conducted across these numerous variables, here we summarize and review the last decade's research covering the role of AQPs in the migration and invasion of cells in diseased states.

BIN1K358R suppresses glial response to plaques in mouse model of Alzheimer's disease.

INTRODUCTION: The BIN1 coding variant rs138047593 (K358R) is linked to Late-Onset Alzheimer's Disease (LOAD) via targeted exome sequencing. METHODS: To elucidate the functional consequences of this rare coding variant on brain amyloidosis and neuroinflammation, we generated BIN1K358R knock-in mice using CRISPR/Cas9 technology. These mice were subsequently bred with 5xFAD transgenic mice, which serve as a model for Alzheimer's pathology. RESULTS: The presence of the BIN1K358R variant leads to increased cerebral amyloid deposition, with a dampened response of astrocytes and oligodendrocytes, but not microglia, at both the cellular and transcriptional levels. This correlates with decreased neurofilament light chain in both plasma and brain tissue. Synaptic densities are significantly increased in both wild-type and 5xFAD backgrounds homozygous for the BIN1K358R variant. DISCUSSION: The BIN1 K358R variant modulates amyloid pathology in 5xFAD mice, attenuates the astrocytic and oligodendrocytic responses to amyloid plaques, decreases damage markers, and elevates synaptic densities. HIGHLIGHTS: BIN1 rs138047593 (K358R) coding variant is associated with increased risk of LOAD. BIN1 K358R variant increases amyloid plaque load in 12-month-old 5xFAD mice. BIN1 K358R variant dampens astrocytic and oligodendrocytic response to plaques. BIN1 K358R variant decreases neuronal damage in 5xFAD mice. BIN1 K358R upregulates synaptic densities and modulates synaptic transmission.

Implementation of Patient Blood Management in Orthotopic Heart Transplants: A Single Centre Retrospective Observational Review.

BACKGROUND: Blood transfusion in the perioperative cardiothoracic setting has accepted risks including deep sternal wound infection, increased intensive care unit length of stay, lung injury, and cost. It has an immunomodulatory effect which may cause allo-immunisation. This may influence long-term survival through immune-mediated factors. Targeting coagulation defects to reduce unnecessary or inappropriate transfusions may reduce these complications. METHODS: In 2012, an institution-wide patient blood management evidence-based algorithmic bleeding management protocol was implemented at The Prince Charles Hospital, Brisbane, Australia. The benefit of this has been previously reported in our lung transplant and cardiac surgery (excluding transplants) cohorts. This study aimed to investigate the effect of this on our orthotopic heart transplant recipients. RESULTS: After the implementation of the protocol, despite no difference in preoperative haemoglobin levels and higher risk patients (EuroSCORE 20 vs 26; p=0.013), the use of packed red blood cells (13.0 U vs 4.4 U; p=0.046) was significantly lower postoperatively and fresh frozen plasma was significantly lower both intra- and postoperatively (7.4 U vs 0.6 U; p<0.001, and 3.3 U vs 0.6 U; p=0.011 respectively). Concurrently, the use of prothrombin complex concentrate (33% vs 78%; p<0.001) and desmopressin (5% vs 22%; p=0.0028) was significantly higher in the post-protocol group, while there was less use of recombinant factor VIIa (15% vs 4%; p=0.058). Intraoperative units of cryoprecipitate also rose from 0.9 to 2.0 (p=0.006). CONCLUSIONS: We have demonstrated that a targeted patient blood management protocol with point-of-care testing for heart transplant recipients is correlated with fewer blood products used postoperatively, with some increase in haemostatic products and no evidence of increased adverse events.

The multifaceted role of aquaporins in physiological cell migration.

Cell migration is an essential process that underlies many physiological processes, including the immune response, organogenesis in the embryo, and angiogenesis, as well as pathological processes such as cancer metastasis. Cells have at their disposal a variety of migratory behaviors and mechanisms that seem to be specific to cell type and the microenvironment. Research over the past two decades has elucidated the water channel protein family of aquaporins (AQPs) as a regulator of many cell migration-related processes, from physical phenomena to biological signaling pathways. The roles that AQPs play in cell migration are both cell type- and isoform-specific; thus, a large swath of information has accumulated as researchers seek to identify the responses across these distinct variables. There does not seem to be a universal role that AQPs play in cell migration; the complex interplay between AQPs and cell volume management, signaling pathway activation, and in a few identified circumstances, gene expression regulation, has shown the intricate, and perhaps paradoxical, role of AQPs in cell migration. The objective of this review is to provide an organized and integrated collection of recent work that has elucidated the many mechanisms by which AQPs regulate cell migration.NEW & NOTEWORTHY Research has elucidated the water channel protein family of aquaporins (AQPs) as a regulator of many cell migration-related processes, from physical phenomena to biological signaling pathways. The roles that AQPs play in cell migration are both cell type- and isoform-specific; thus, a large swath of information has accumulated as researchers seek to identify the responses across these distinct variables. This review compiles insights into the recent findings linking AQPs to physiological cell migration.

Nephrotoxicity of Vancomycin in Combination With Beta-Lactam Agents: Ceftolozane-Tazobactam vs Piperacillin-Tazobactam.

BACKGROUND: Vancomycin (VAN)-associated acute kidney injury (AKI) is increased when VAN is combined with certain beta-lactams (BLs) such as piperacillin-tazobactam (TZP) but has not been evaluated with ceftolozane-tazobactam (C/T). Our aim was to investigate the AKI incidence of VAN in combination with C/T (VAN/C/T) compared with VAN in combination to TZP (VAN-TZP). METHODS: We conducted a multicenter, observational, comparative study across the United States. The primary analysis was a composite outcome of AKI and risk, injury, failure, loss, end stage renal disease; Acute Kidney Injury Network; or VAN-induced nephrotoxicity according to the consensus guidelines. Multivariable logistic regression analysis was conducted to adjust for confounding variables and stratified Kaplan-Meir analysis to assess the time to nephrotoxicity between the 2 groups. RESULTS: We included VAN/C/T (n = 90) and VAN-TZP (n = 284) at an enrollment ratio of 3:1. The primary outcome occurred in 12.2% vs 25.0% in the VAN-C/T and VAN-TZP groups, respectively (P = .011). After adjusting for confounding variables, VAN-TZP was associated with increased odds of AKI compared with VAN-C/T; with an adjusted odds ratio of 3.308 (95% confidence interval, 1.560-6.993). Results of the stratified Kaplan-Meir analysis with log-rank time-to-nephrotoxicity analysis indicate that time to AKI was significantly shorter among patients who received VAN-TZP (P = .004). Cox proportional hazards analysis demonstrated that TZP was consistent with the primary analysis (P = .001). CONCLUSIONS: Collectively, our results suggest that the AKI is not likely to be related to tazobactam but rather to piperacillin, which is a component in VAN-TZP but not in VAN-C/T.

Clinico-pathologic relationships with Ki67 and its change with short-term aromatase inhibitor treatment in primary ER + breast cancer: further results from the POETIC trial (CRUK/07/015).

PURPOSE: Ki67 assessed at diagnosis (Ki67baseline) is an important prognostic factor in primary oestrogen receptor-positive (ER +) breast cancer. Proportional change in Ki67 after 2 weeks (∆Ki672week) is associated with clinical benefit from endocrine therapies and residual Ki67 (Ki672week) with recurrence-free survival. The aim was to define the association between Ki67baseline and after aromatase inhibitor (AI) exposure ∆Ki672week and Ki672week with key prognostic and biologic factors utilising data from the POETIC study. PATIENTS AND METHODS: In POETIC 4480 postmenopausal patients with primary ER and/or PgR + breast cancer were randomised 2:1 to 2 weeks' presurgical AI (anastrozole or letrozole) or no presurgical treatment (control). Ki67 was measured centrally in core-cut biopsies taken prior to AI and in core-cuts or the excision biopsy at surgery. Relationships between the Ki67 and biologic factors were explored using linear regression. RESULTS: Established associations of Ki67baseline with biologic factors including PgR status, tumour grade, tumour size, histological subtype, nodal status, and vascular invasion were confirmed in the HER2- subpopulation. In the HER2 + subpopulation only grade and tumour size were significantly associated with Ki67baseline. In control group Ki672week was 18% lower than Ki67baseline (p < 0.001) when Ki672week was measured in excision biopsies but not when measured in core-cuts. Median suppression by AIs (∆Ki672week) was 79.3% (IQR: -89.9 to -54.6) and 53.7% (IQR: -78.9 to -21.1) for HER2-negative and HER2-positive cases, respectively. Significantly less suppression occurred in PgR- vs PgR + and HER2 + vs HER2- tumours which remained apparent after adjustment for 2-week sample type. CONCLUSIONS: The magnitude of this study allowed characterisation of relationships between Ki67baseline, ∆Ki672week and Ki672week with high degrees of confidence providing a reference source for other studies. Lower values of Ki67 occur when measured on excision biopsies and could lead to apparent but artefactual decreases in Ki67: this should be considered when either ∆Ki672week or Ki672week is used in routine clinical practice to aid treatment decisions or in clinical trials assessing new drug therapies.

Structure of blood coagulation factor VIII in complex with an anti-C1 domain pathogenic antibody inhibitor.

Antibody inhibitor development in hemophilia A represents the most significant complication resulting from factor VIII (fVIII) replacement therapy. Recent studies have demonstrated that epitopes present in the C1 domain contribute to a pathogenic inhibitor response. In this study, we report the structure of a group A anti-C1 domain inhibitor, termed 2A9, in complex with a B domain-deleted, bioengineered fVIII construct (ET3i). The 2A9 epitope forms direct contacts to the C1 domain at 3 different surface loops consisting of Lys2065-Trp2070, Arg2150-Tyr2156, and Lys2110-Trp2112. Additional contacts are observed between 2A9 and the A3 domain, including the Phe1743-Tyr1748 loop and the N-linked glycosylation at Asn1810. Most of the C1 domain loops in the 2A9 epitope also represent a putative interface between fVIII and von Willebrand factor. Lastly, the C2 domain in the ET3i:2A9 complex adopts a large, novel conformational change, translocating outward from the structure of fVIII by 20 Å. This study reports the first structure of an anti-C1 domain antibody inhibitor and the first fVIII:inhibitor complex with a therapeutically active fVIII construct. Further structural understanding of fVIII immunogenicity may result in the development of more effective and safe fVIII replacement therapies.

Comparisons on factors affecting residents fulfilling self-determination in ethno-specific and mainstream nursing homes: a qualitative study.

BACKGROUND: Studies revealed that supporting residents fulfilling self-determination is positively associated with their health, wellbeing and quality of life. Cross-cultural care poses significant challenges for nursing home residents to fulfil their self-determination in control of own care and maintaining meaningful connections with others. The aim of the study was to compare factors affecting residents fulfilling self-determination in ethno-specific and mainstream nursing homes. METHODS: A qualitative descriptive approach was applied to the study. Culturally competent care and person-centred care were employed as guiding frameworks. Individual interviews or a focus group with residents and family members were conducted to collect data. RESULTS: In total, 29 participants participated in the study. Three main themes were identified: communicating needs and preferences; mastering own care; and maintaining meaningful relationships. Each theme includes sub-themes that detail similarities and differences of factors affecting residents fulfilling self-determination in the two type nursing homes. Findings indicate that residents from both types of nursing homes experienced challenges to communicate their care needs and preferences in daily care activities. Moreover, residents or their representatives from both types of nursing homes demonstrated motivation and competence to master residents' care based on their individual preferences, but also perceived that their motivation was not always supported by staff or the nursing home environment. Residents' competence in mastering their care activities in ethno-specific nursing homes was based on the condition that they were given opportunities to use a language of choice in communication and staff and the nursing home demonstrated culturally competent care for them. In addition, ethno-specific nursing homes showed more recourse to support residents to maintain meaningful relationships with peers and others. CONCLUSIONS: Culturally competent care created by staff, nursing homes and the aged care system is a basic condition for residents from ethnic minority groups to fulfil self-determination. In addition, person-centred care approach enables residents to optimise self-determination.

Characterization of the Biophysical Properties and Cell Adhesion Interactions of Marine Invertebrate Collagen from Rhizostoma pulmo.

Collagen is the most ubiquitous biomacromolecule found in the animal kingdom and is commonly used as a biomaterial in regenerative medicine therapies and biomedical research. The collagens used in these applications are typically derived from mammalian sources which poses sociological issues due to widespread religious constraints, rising ethical concern over animal rights and the continuous risk of zoonotic disease transmission. These issues have led to increasing research into alternative collagen sources, of which marine collagens, in particular from jellyfish, have emerged as a promising resource. This study provides a characterization of the biophysical properties and cell adhesion interactions of collagen derived from the jellyfish Rhizostoma pulmo (JCol). Circular dichroism spectroscopy and atomic force microscopy were used to observe the triple-helical conformation and fibrillar morphology of JCol. Heparin-affinity chromatography was also used to demonstrate the ability of JCol to bind to immobilized heparin. Cell adhesion assays using integrin blocking antibodies and HT-1080 human fibrosarcoma cells revealed that adhesion to JCol is primarily performed via ß1 integrins, with the exception of α2ß1 integrin. It was also shown that heparan sulfate binding plays a much greater role in fibroblast and mesenchymal stromal cell adhesion to JCol than for type I mammalian collagen (rat tail collagen). Overall, this study highlights the similarities and differences between collagens from mammalian and jellyfish origins, which should be considered when utilizing alternative collagen sources for biomedical research.

TRM integrins CD103 and CD49a differentially support adherence and motility after resolution of influenza virus infection.

Tissue-resident memory CD8 T (TRM) cells are a unique immune memory subset that develops and remains in peripheral tissues at the site of infection, providing future host resistance upon reexposure to that pathogen. In the pulmonary system, TRM are identified through S1P antagonist CD69 and expression of integrins CD103/ß7 and CD49a/CD29(ß1). Contrary to the established role of CD69 on CD8 T cells, the functions of CD103 and CD49a on this population are not well defined. This study examines the expression patterns and functions of CD103 and CD49a with a specific focus on their impact on T cell motility during influenza virus infection. We show that the TRM cell surface phenotype develops by 2 wk postinfection, with the majority of the population expressing CD49a and a subset that is also positive for CD103. Despite a previously established role in retaining TRM in peripheral tissues, CD49a facilitates locomotion of virus-specific CD8 T cells, both in vitro and in vivo. These results demonstrate that CD49a may contribute to local surveillance mechanisms of the TRM population.

Staff perceived challenges and facilitators in supporting resident self-determination in ethno-specific and mainstream nursing homes.

AIMS AND OBJECTIVES: To explore and compare staff perceived challenges and facilitators in supporting resident self-determination in ethno-specific and mainstream nursing homes. BACKGROUND: Staff and residents in ethno-specific and mainstream nursing homes in most developed countries have shown increased cultural and linguistic diversity. This socio-demographic change poses significant challenges for staff to support resident self-determination of their own care. In-depth understanding of those challenges in the two types of nursing homes is much needed to inform practice in nurse-led nursing home care settings. METHOD: A qualitative description approach with thematic analysis was used in the study. Data were collected through five focus groups with 29 various direct care workers from two ethno-specific nursing homes and a mainstream nursing home in Australia between March-September 2020. The study report followed the COREQ checklist. RESULTS: Four themes were identified from focus group data. First, participants perceived communication challenges in identifying residents' preferences, especially in ethno-specific nursing homes. Second, team efforts that included residents and their family members were highly valued as a way to meet residents' preferences. Third, participants described various levels of staff engagement in residents' care planning. In addition, staff in ethno-specific nursing homes possessed richer resources to maintain meaningful relationships for residents compared with their counterparts in the mainstream nursing home. CONCLUSIONS: Staff in ethno-specific nursing homes experience more challenges in supporting resident self-determination but have richer resources to develop culturally safe and culturally competent care compared with their counterparts in the mainstream nursing home. RELEVANCE TO CLINICAL PRACTICE: Findings provide new insights into challenges and practical solutions in supporting residents to self-determine their own care in cross-cultural aged care. PATIENT OR PUBLIC CONTRIBUTION: This study was co-designed with three aged care organisations who funded the study. Staff employed by these organisations participated in the study.

IsobaricQuant enables cross-platform quantification, visualization, and filtering of isobarically-labeled peptides.

In mass spectrometry (MS)-based quantitative proteomics, labeling with isobaric mass tags such as iTRAQ and TMT can substantially improve sample throughput and reduce peptide missing values. Nonetheless, the quantification of labeled peptides tends to suffer from reduced accuracy due to the co-isolation of co-eluting precursors of similar mass-to-charge. Acquisition approaches such as multistage MS3 or ion mobility separation address this problem, yet are difficult to audit and limited to expensive instrumentation. Here we introduce IsobaricQuant, an open-source software tool for quantification, visualization, and filtering of peptides labeled with isobaric mass tags, with specific focus on precursor interference. IsobaricQuant is compatible with MS2 and MS3 acquisition strategies, has a viewer that allows assessing interference, and provides several scores to aid the filtering of scans with compression. We demonstrate that IsobaricQuant quantifications are accurate by comparing it with commonly used software. We further show that its QC scores can successfully filter out scans with reduced quantitative accuracy at MS2 and MS3 levels, removing inaccurate peptide quantifications and decreasing protein CVs. Finally, we apply IsobaricQuant to a PISA dataset and show that QC scores improve the sensitivity of the identification of protein targets of a kinase inhibitor. IsobaricQuant is available at https://github.com/Villen-Lab/isobaricquant.

Evaluation of statistical approaches for association testing in noisy drug screening data.

BACKGROUND: Identifying associations among biological variables is a major challenge in modern quantitative biological research, particularly given the systemic and statistical noise endemic to biological systems. Drug sensitivity data has proven to be a particularly challenging field for identifying associations to inform patient treatment. RESULTS: To address this, we introduce two semi-parametric variations on the commonly used concordance index: the robust concordance index and the kernelized concordance index (rCI, kCI), which incorporate measurements about the noise distribution from the data. We demonstrate that common statistical tests applied to the concordance index and its variations fail to control for false positives, and introduce efficient implementations to compute p-values using adaptive permutation testing. We then evaluate the statistical power of these coefficients under simulation and compare with Pearson and Spearman correlation coefficients. Finally, we evaluate the various statistics in matching drugs across pharmacogenomic datasets. CONCLUSIONS: We observe that the rCI and kCI are better powered than the concordance index in simulation and show some improvement on real data. Surprisingly, we observe that the Pearson correlation was the most robust to measurement noise among the different metrics.

Methods for isolating and analyzing physiological hyaluronan: a review.

The carbohydrate hyaluronan (or hyaluronic acid, HA) is found in all human tissues and biofluids where it has wide-ranging functions in health and disease that are dictated by both its abundance and size. Consequently, hyaluronan evaluation in physiological samples has significant translational potential. Although the analytical tools and techniques for probing other biomolecules such as proteins and nucleic acids have become standard approaches in biochemistry, those available for investigating hyaluronan are less well established. In this review, we survey methods related to the assessment of native hyaluronan in biological specimens, including protocols for separating it from biological matrices and technologies for determining its concentration and molecular weight.

Relationship between ER expression by IHC or mRNA with Ki67 response to aromatase inhibition: a POETIC study.

BACKGROUND: In clinical practice, oestrogen receptor (ER) analysis is almost entirely by immunohistochemistry (IHC). ASCO/CAP recommends cut-offs of < 1% (negative) and 1-10% (low) cells positive. There is uncertainty whether patients with ER low tumours benefit from endocrine therapy. We aimed to assess IHC and mRNA cut-points for ER versus biological response of primary breast cancer to 2 weeks' aromatase inhibitor treatment as measured by change in Ki67. METHODS: Cases were selected from the aromatase inhibitor treatment group of POETIC. We selected the 15% with the poorest Ki67 response (PR, < 40% Ki67 suppression, n = 230) and a random 30% of the remainder categorised as intermediate (IR, 40-79% Ki67 suppression, n = 150) and good-responders (GR, ≥ 80% Ki67 suppression, n = 230) from HER2 - group. All HER2 + cases available were selected irrespective of their response category (n = 317). ER expression was measured by IHC and qPCR. RESULTS: ER IHC was available from 515 HER2 - and 186 HER2 + tumours and ER qPCR from 367 HER2 - and 171 HER2 + tumours. Ninety-one percentage of patients with ER IHC < 10% were PRs with similar rates in HER2 - and HER2 + cases. At or above ER IHC 10% substantial numbers of patients showed IR or GR. Similar proportions of patients were defined by cut-points of ER IHC < 10% and ER mRNA < 5 units. In addition, loss of PgR expression altered ER anti-proliferation response with 92% of PgR - cases with ER IHC < 40% being PRs. CONCLUSIONS: There was little responsiveness at IHC < 10% and no distinction between < 1% and 1-10% cells positive. Similar separation of PRs from IR/GRs was achieved by IHC and mRNA.

Coisolation of Peptide Pairs for Peptide Identification and MS/MS-Based Quantification.

Stable-isotope labeling with amino acids in cell culture (SILAC)-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data-dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a DDA strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. This peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions. This study shows the feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification.