Death-associated protein kinase 3 modulates migration and invasion of triple-negative breast cancer cells.

Sixteen patient-derived xenografts (PDXs) were analyzed using a mass spectrometry (MS)-based kinase inhibitor pull-down assay (KIPA), leading to the observation that death-associated protein kinase 3 (DAPK3) is significantly and specifically overexpressed in the triple-negative breast cancer (TNBC) models. Validation studies confirmed enrichment of DAPK3 protein, in both TNBC cell lines and tumors, independent of mRNA levels. Genomic knockout of DAPK3 in TNBC cell lines inhibited in vitro migration and invasion, along with down-regulation of an epithelial-mesenchymal transition (EMT) signature, which was confirmed in vivo. The kinase and leucine-zipper domains within DAPK3 were shown by a mutational analysis to be essential for functionality. Notably, DAPK3 was found to inhibit the levels of desmoplakin (DSP), a crucial component of the desmosome complex, thereby explaining the observed migration and invasion effects. Further exploration with immunoprecipitation-mass spectrometry (IP-MS) identified that leucine-zipper protein 1 (LUZP1) is a preferential binding partner of DAPK3. LUZP1 engages in a leucine-zipper domain-mediated interaction that protects DAPK3 from proteasomal degradation. Thus, the DAPK3/LUZP1 heterodimer emerges as a newly discovered regulator of EMT/desmosome components that promote TNBC cell migration.

Association of Serum Biomarkers With Neurocognitive Decline After PCI in Small Cell Lung Cancer: An Exploratory Study of the Phase III NCT01780675 Trial.

INTRODUCTION: Blood samples were collected to explore potential serum biomarkers associated with neurocognitive function in small-cell lung cancer (SCLC) patients who received prophylactic cranial irradiation (PCI). METHODS: This pre-specified study included patients with blood samples available, who participated in a phase III trial (NCT01780675). Blood samples were collected before PCI and 3-days post-initiating PCI. Neurocognitive decline was defined as a decrease of ≥ 5 points on total recall in the Hopkins Verbal Learning Test-Revised (HVLT-R) assessed from pre-PCI to 4-months post-PCI. Biomarkers were screened using univariate logistic regression analysis. P < .1 was considered statistically significant. RESULTS: Forty-eight enrolled patients who had blood samples at baseline were included and 27 were available for analysis as the other 21 did not assess neurocognitive function at 4-months. Lower levels of Tie-2 (OR = 0.999, 90% CI 0.998-1.000, P = .062), and higher levels of MIP-1b (OR = 1.022, 90% CI 1.000-1.044, P = .093), CCL-17 (OR = 1.004, 90% CI 1.001-1.006, P = .029), and IL-1α (OR = 1.597, 90% CI 1.077-2.367, P = .05) before PCI were correlated with neurocognitive decline at 4-months. Decrease of VEGF-C (OR = 0.972, 90% CI 0.949-0.996, P = .055), CCL-17 (OR = 0.993, 90% CI 0.988-0.999, P = .036), IL-1α (OR = 0.788, 90% CI 0.635-0.979, P = .071), and VEGF (OR = 0.981, 90% CI 0.965-0.997, P = .051) 3-days post-initiating PCI were also associated with neurocognitive decline at 4-months. CONCLUSIONS: Biomarker levels before PCI and changes in their levels 3-days post-initiating PCI may be linked to subsequent neurocognitive decline at 4-months. If validated, these biomarkers could be used to predict the risk of neurocognitive decline and act as a decision aid for personalized PCI in SCLC.

The 100 highest-cited original articles in large vessel occlusions: A bibliometric analysis.

BACKGROUND: Large vessel occlusions (LVO) are a common etiology of morbidity and mortality. The current literature lacks a synthesis of the landscape and trends in research. OBJECTIVE: We aimed to conduct a bibliometric analysis of the 100 most cited original articles on LVOs to assess the current state of research. METHODS: Scopus database was queried from inception to December 2022 to identify the most cited original articles from 4506 retrieved records on LVOs. Publication year, country of origin, total and average annual citation count, and type of study were collected for each article. The journal impact factor (JIF) was obtained from the Journal Citation Reports database. RESULTS: The articles were published between 1994 and 2021, with most (n = 82) published during the 2011-2020 decade. The median total citation count was 108.5, with an interquartile range (IQR) of 81-149.5. The median (IQR) average annual citation count was 15.9 (11.5-22.9). Half of the articles were published in Stroke (n = 35) and Journal of NeuroInterventional Surgery (n = 15), with JIFs ranging from 1.8 to 202.7. The USA was the leading country in contributing to LVO research (n = 45). Most studies focused on the treatment (n = 63) and diagnosis (n = 22) of LVOs. CONCLUSIONS: Most articles were published during the past decade, highlighting the impact of the clinical trials of endovascular treatment on the discipline. With several ongoing clinical trials on the horizon, continued growth of the field is anticipated in the upcoming decades.

The astrocyte α1A-adrenoreceptor is a key component of the neuromodulatory system in mouse visual cortex.

Noradrenaline (norepinephrine) is known to modulate many physiological functions and behaviors. In this study, we tested to what extent astrocytes, a type of glial cell, participate in noradrenergic signaling in mouse primary visual cortex (V1). Astrocytes are essential partners of neurons in the central nervous system. They are central to brain homeostasis, but also dynamically regulate neuronal activity, notably by relaying and regulating neuromodulator signaling. Indeed, astrocytes express receptors for multiple neuromodulators, including noradrenaline, but the extent to which astrocytes are involved in noradrenergic signaling remains unclear. To test whether astrocytes are involved in noradrenergic neuromodulation in mice, we employed both short hairpin RNA mediated knockdown as well as pharmacological manipulation of the major noradrenaline receptor in astrocytes, the α1A-adrenoreceptor. Using acute brain slices, we found that the astrocytic α1A-adrenoreceptor subtype contributes to the generation of large intracellular Ca2+ signals in visual cortex astrocytes, which are generally thought to underlie astrocyte function. To test if reduced α1A-adrenoreceptor signaling in astrocytes affected the function of neuronal circuits in V1, we used both patch-clamp and field potential recordings. These revealed that noradrenergic signaling through the astrocyte α1A-adrenoreceptor is important to not only modulate synaptic activity but also to regulate plasticity in V1, through the potentiation of synaptic responses in circuits involved in visual information processing.

Regulatory T cells as a possible new target in epilepsy?

Epilepsy is a complex chronic brain disorder with diverse clinical features that can be caused by various triggering events, such as infections, head trauma, or stroke. During epileptogenesis, various abnormalities are observed, such as altered cellular homeostasis, imbalance of neurotransmitters, tissue changes, and the release of inflammatory mediators, which in combination lead to spontaneous recurrent seizures. Regulatory T cells (Tregs), a subtype of CD4+Foxp3+ T cells, best known for their key function in immune suppression, also seem to play a role in attenuating neurodegeneration and suppressing pathological inflammation in several brain disease states. Considering that epilepsy is also highly associated with neuronal damage and neuroinflammation, modulation of Tregs may be an interesting way to modify the disease course of epilepsy and needs further investigation. In this review, we will describe the currently available information on Tregs in epilepsy.

Do we really apply evidence-based-recommendations to spine surgery? Results of an international survey.

OBJECTIVE: This international survey investigated Evidence-Based Medicine (EBM) in spine surgery by measuring its acceptance among spine surgeons. It assessed their understanding of EBM and how they apply it in practice by analyzing responses to various clinical scenarios.. MATERIALS AND METHODS: Following the CHERRIES guidelines, an e-survey was distributed to multiple social media forums for neurosurgeons and orthopedic surgeons on Facebook, LinkedIn, and Telegram and circulated further through email via the authors' network. Three hundred participants from Africa, Asia, Europe, North America, and Oceania completed the survey. RESULTS: Our study revealed that 67.7% (n = 203) of respondents used EBM in their practice, and 97.3% (n = 292) believed training in research methodology and EBM was necessary for the practice of spine surgery. Despite this endorsement of using EBM in spine surgery, we observed varied responses to how EBM is applied in practice based on example scenarios. The responders who had additional training tended to obey EBM guidelines more than those who had no additional training. Most surgeons responded as always or sometimes prescribing methylprednisolone to patients with acute spinal cord injury. Other significant differences were identified between geographical regions, training, practice settings, and other factors. CONCLUSIONS: Most respondents used EBM in practice and believed training in research methodology and EBM is necessary for spine surgery; however, there were significant variations on how to use them per case. Thus, the appropriate application of EBM in clinical settings for spinal surgery must be further studied.

PKMYT1 Is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER+ Breast Cancer.

Endocrine therapies (ET) with cyclin-dependent kinase 4/6 (CDK4/6) inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of patient-derived xenografts (PDXs) from patients with 22 ER+ breast cancer demonstrated that protein kinase, membrane-associated tyrosine/threonine one (PKMYT1), a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX-derived organoids and PDXs, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.

Kinase inhibitor pulldown assay (KiP) for clinical proteomics.

Protein kinases are frequently dysregulated and/or mutated in cancer and represent essential targets for therapy. Accurate quantification is essential. For breast cancer treatment, the identification and quantification of the protein kinase ERBB2 is critical for therapeutic decisions. While immunohistochemistry (IHC) is the current clinical diagnostic approach, it is only semiquantitative. Mass spectrometry-based proteomics offers quantitative assays that, unlike IHC, can be used to accurately evaluate hundreds of kinases simultaneously. The enrichment of less abundant kinase targets for quantification, along with depletion of interfering proteins, improves sensitivity and thus promotes more effective downstream analyses. Multiple kinase inhibitors were therefore deployed as a capture matrix for kinase inhibitor pulldown (KiP) assays designed to profile the human protein kinome as broadly as possible. Optimized assays were initially evaluated in 16 patient derived xenograft models (PDX) where KiP identified multiple differentially expressed and biologically relevant kinases. From these analyses, an optimized single-shot parallel reaction monitoring (PRM) method was developed to improve quantitative fidelity. The PRM KiP approach was then reapplied to low quantities of proteins typical of yields from core needle biopsies of human cancers. The initial prototype targeting 100 kinases recapitulated intrinsic subtyping of PDX models obtained from comprehensive proteomic and transcriptomic profiling. Luminal and HER2 enriched OCT-frozen patient biopsies subsequently analyzed through KiP-PRM also clustered by subtype. Finally, stable isotope labeled peptide standards were developed to define a prototype clinical method. Data are available via ProteomeXchange with identifiers PXD044655 and PXD046169.

Unravelling cell type-specific responses to Parkinson's Disease at single cell resolution.

Parkinson's Disease (PD) is the second most common neurodegenerative disorder. The pathological hallmark of PD is loss of dopaminergic neurons and the presence of aggregated α-synuclein, primarily in the substantia nigra pars compacta (SNpc) of the midbrain. However, the molecular mechanisms that underlie the pathology in different cell types is not currently understood. Here, we present a single nucleus transcriptome analysis of human post-mortem SNpc obtained from 15 sporadic Parkinson's Disease (PD) cases and 14 Controls. Our dataset comprises ∼84K nuclei, representing all major cell types of the brain, allowing us to obtain a transcriptome-level characterization of these cell types. Importantly, we identify multiple subpopulations for each cell type and describe specific gene sets that provide insights into the differing roles of these subpopulations. Our findings reveal a significant decrease in neuronal cells in PD samples, accompanied by an increase in glial cells and T cells. Subpopulation analyses demonstrate a significant depletion of tyrosine hydroxylase (TH) enriched astrocyte, microglia and oligodendrocyte populations in PD samples, as well as TH enriched neurons, which are also depleted. Moreover, marker gene analysis of the depleted subpopulations identified 28 overlapping genes, including those associated with dopamine metabolism (e.g., ALDH1A1, SLC6A3 & SLC18A2). Overall, our study provides a valuable resource for understanding the molecular mechanisms involved in dopaminergic neuron degeneration and glial responses in PD, highlighting the existence of novel subpopulations and cell type-specific gene sets.

Intracortical brain-computer interfaces for improved motor function: a systematic review.

In this systematic review, we address the status of intracortical brain-computer interfaces (iBCIs) applied to the motor cortex to improve function in patients with impaired motor ability. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 Guidelines for Systematic Reviews. Risk Of Bias In Non-randomized Studies - of Interventions (ROBINS-I) and the Effective Public Health Practice Project (EPHPP) were used to assess bias and quality. Advances in iBCIs in the last two decades demonstrated the use of iBCI to activate limbs for functional tasks, achieve neural typing for communication, and other applications. However, the inconsistency of performance metrics employed by these studies suggests the need for standardization. Each study was a pilot clinical trial consisting of 1-4, majority male (64.28 %) participants, with most trials featuring participants treated for more than 12 months (55.55 %). The systems treated patients with various conditions: amyotrophic lateral sclerosis, stroke, spinocerebellar degeneration without cerebellar involvement, and spinal cord injury. All participants presented with tetraplegia at implantation and were implanted with microelectrode arrays via pneumatic insertion, with nearly all electrode locations solely at the precentral gyrus of the motor cortex (88.88 %). The development of iBCI devices using neural signals from the motor cortex to improve motor-impaired patients has enhanced the ability of these systems to return ability to their users. However, many milestones remain before these devices can prove their feasibility for recovery. This review summarizes the achievements and shortfalls of these systems and their respective trials.

Treatment outcomes of complement protein C5 inhibition in 509 UK patients with paroxysmal nocturnal hemoglobinuria.

ABSTRACT: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic disorder that occurs on a background of bone marrow failure (BMF). In PNH, chronic intravascular hemolysis causes an increase in morbidity and mortality, mainly because of thromboses. Over the last 20 years, treatment of PNH has focused on the complement protein C5 to prevent intravascular hemolysis using the monoclonal antibody eculizumab and more recently ravulizumab. In the United Kingdom, all patients are under review at 1 of 2 reference centers. We report on all 509 UK patients with PNH treated with eculizumab and/or ravulizumab between May 2002 and July 2022. The survival of patients with eculizumab and ravulizumab was significantly lower than that of age- and sex-matched controls (P = .001). Only 4 patients died of thromboses. The survival of patients with PNH (n = 389), when those requiring treatment for BMF (clonal evolution to myelodysplastic syndrome or acute leukemia or had progressive unresponsive aplastic anemia) were excluded, was not significantly different from that of age- and sex-matched controls (P = .12). There were 11 cases of meningococcal sepsis (0.35 events per 100 patient-years). Extravascular hemolysis was evident in patients who received treatment, with 26.7% of patients requiring transfusions in the most recent 12 months on therapy. Eculizumab and ravulizumab are safe and effective therapies that reduce mortality and morbidity in PNH, but further work is needed to reduce mortality in those with concomitant BMF.

Chemogenetic manipulation of astrocyte activity at the synapse- a gateway to manage brain disease.

Astrocytes are the major glial cell type in the central nervous system (CNS). Initially regarded as supportive cells, it is now recognized that this highly heterogeneous cell population is an indispensable modulator of brain development and function. Astrocytes secrete neuroactive molecules that regulate synapse formation and maturation. They also express hundreds of G protein-coupled receptors (GPCRs) that, once activated by neurotransmitters, trigger intracellular signalling pathways that can trigger the release of gliotransmitters which, in turn, modulate synaptic transmission and neuroplasticity. Considering this, it is not surprising that astrocytic dysfunction, leading to synaptic impairment, is consistently described as a factor in brain diseases, whether they emerge early or late in life due to genetic or environmental factors. Here, we provide an overview of the literature showing that activation of genetically engineered GPCRs, known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to specifically modulate astrocyte activity partially mimics endogenous signalling pathways in astrocytes and improves neuronal function and behavior in normal animals and disease models. Therefore, we propose that expressing these genetically engineered GPCRs in astrocytes could be a promising strategy to explore (new) signalling pathways which can be used to manage brain disorders. The precise molecular, functional and behavioral effects of this type of manipulation, however, differ depending on the DREADD receptor used, targeted brain region and timing of the intervention, between healthy and disease conditions. This is likely a reflection of regional and disease/disease progression-associated astrocyte heterogeneity. Therefore, a thorough investigation of the effects of such astrocyte manipulation(s) must be conducted considering the specific cellular and molecular environment characteristic of each disease and disease stage before this has therapeutic applicability.

Kinase Inhibitor Pulldown Assay Identifies a Chemotherapy Response Signature in Triple-negative Breast Cancer Based on Purine-binding Proteins.

Triple-negative breast cancer (TNBC) constitutes 10%-15% of all breast tumors. The current standard of care is multiagent chemotherapy, which is effective in only a subset of patients. The original objective of this study was to deploy a mass spectrometry (MS)-based kinase inhibitor pulldown assay (KIPA) to identify kinases elevated in non-pCR (pathologic complete response) cases for therapeutic targeting. Frozen optimal cutting temperature compound-embedded core needle biopsies were obtained from 43 patients with TNBC before docetaxel- and carboplatin-based neoadjuvant chemotherapy. KIPA was applied to the native tumor lysates that were extracted from samples with high tumor content. Seven percent of all identified proteins were kinases, and none were significantly associated with lack of pCR. However, among a large population of "off-target" purine-binding proteins (PBP) identified, seven were enriched in pCR-associated samples (P < 0.01). In orthogonal mRNA-based TNBC datasets, this seven-gene "PBP signature" was associated with chemotherapy sensitivity and favorable clinical outcomes. Functional annotation demonstrated IFN gamma response, nuclear import of DNA repair proteins, and cell death associations. Comparisons with standard tandem mass tagged-based discovery proteomics performed on the same samples demonstrated that KIPA-nominated pCR biomarkers were unique to the platform. KIPA is a novel biomarker discovery tool with unexpected utility for the identification of PBPs related to cytotoxic drug response. The PBP signature has the potential to contribute to clinical trials designed to either escalate or de-escalate therapy based on pCR probability. Significance: The identification of pretreatment predictive biomarkers for pCR in response to neoadjuvant chemotherapy would advance precision treatment for TNBC. To complement standard proteogenomic discovery profiling, a KIPA was deployed and unexpectedly identified a seven-member non-kinase PBP pCR-associated signature. Individual members served diverse pathways including IFN gamma response, nuclear import of DNA repair proteins, and cell death.

Proteogenomic Approaches for the Identification of NF1/Neurofibromin-depleted Estrogen Receptor-positive Breast Cancers for Targeted Treatment.

NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader, together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ patient-derived xenograft (PDX) models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pulldown. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An IHC assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Significance: A major challenge for targeting the consequence of tumor suppressor disruption is the accurate assessment of protein functional inactivation. NF1 can repress both RAS and ER signaling, and a ComboMATCH trial is underway to treat the patients with binimetinib and fulvestrant. Herein we report a MS-verified NF1 IHC assay that can determine a threshold for NF1 loss to predict treatment response. These approaches may be used to identify and expand the eligible patient population.

The Breast Cancer Proteome and Precision Oncology.

The goal of precision oncology is to translate the molecular features of cancer into predictive and prognostic tests that can be used to individualize treatment leading to improved outcomes and decreased toxicity. Success for this strategy in breast cancer is exemplified by efficacy of trastuzumab in tumors overexpressing ERBB2 and endocrine therapy for tumors that are estrogen receptor positive. However, other effective treatments, including chemotherapy, immune checkpoint inhibitors, and CDK4/6 inhibitors are not associated with strong predictive biomarkers. Proteomics promises another tier of information that, when added to genomic and transcriptomic features (proteogenomics), may create new opportunities to improve both treatment precision and therapeutic hypotheses. Here, we review both mass spectrometry-based and antibody-dependent proteomics as complementary approaches. We highlight how these methods have contributed toward a more complete understanding of breast cancer and describe the potential to guide diagnosis and treatment more accurately.