Nir1-LNS2 is a novel phosphatidic acid biosensor that reveals mechanisms of lipid production.

Despite various roles of phosphatidic acid (PA) in cellular functions such as lipid homeostasis and vesicular trafficking, there is a lack of high-affinity tools to study PA in live cells. After analysis of the predicted structure of the LNS2 domain in the lipid transfer protein Nir1, we suspected that this domain could serve as a novel PA biosensor. We created a fluorescently tagged Nir1-LNS2 construct and then performed liposome binding assays as well as pharmacological and genetic manipulations of HEK293A cells to determine how specific lipids affect the interaction of Nir1-LNS2 with membranes. We found that Nir1-LNS2 bound to both PA and PIP2 in vitro. Interestingly, only PA was necessary and sufficient to localize Nir1-LNS2 to membranes in cells. Nir1-LNS2 also showed a heightened responsiveness to PA when compared to biosensors using the Spo20 PA binding domain (PABD). Nir1-LNS2's high sensitivity revealed a modest but discernible contribution of PLD to PA production downstream of muscarinic receptors, which has not been visualized with previous Spo20-based probes. In summary, Nir1-LNS2 emerges as a versatile and sensitive biosensor, offering researchers a new powerful tool for real-time investigation of PA dynamics in live cells.

Tunable fluorescent probes for detecting aldehydes in living systems.

Aldehydes, pervasive in various environments, pose health risks at elevated levels due to their collective toxic effects via shared mechanisms. Monitoring total aldehyde content in living systems is crucial due to their cumulative impact. Current methods for detecting cellular aldehydes are limited to UV and visible ranges, restricting their analysis in living systems. This study introduces an innovative reaction-based trigger that leverages the exceptional selectivity of 2-aminothiophenol for aldehydes, leading to the production of dihydrobenzothiazole and activating a fluorescence response. Using this trigger, we developed a series of fluorescent probes for aldehydes by altering the fluorophore allowing for excitation and emission wavelengths across the visible to near-infrared spectral regions without compromising the reactivity of the bioorthogonal moiety. These probes exhibit remarkable aldehyde chemoselectivity, rapid kinetics, and high quantum yields, enabling the detection of diverse aldehyde types, both exogenous and endogenous, within complex biological contexts. Notably, we employed the most red-shifted near-infrared probe from this series to detect aldehydes in living systems, including biliary organoids and mouse organs. These probes provide valuable tools for exploring the multifaceted roles of aldehydes in biological functions and diseases within living systems, laying the groundwork for further investigations.

Patient-targeted education (ePRO-E) to increase ePRO intent within an Alliance clinical trial (A221805-SI1).

BACKGROUND: The Patient Cloud ePRO app was adopted by the National Cancer Institute National Clinical Trials Network (NCTN) to facilitate capturing electronic patient-reported (ePRO) outcome data, but use has been low. The study objectives were to test whether a patient-targeted ePRO educational resource (ePRO-E) would increase ePRO intent (number of users) and improve data quality (high quality: ≥80% of the required surveys submitted) within an ongoing NCTN study. METHODS: The ePRO-E intervention, a patient-targeted educational resource (written material and 6-minute animated YouTube video), was designed to address ePRO barriers. ePRO intent and data quality were compared between 2 groups (N = 69): a historical control group and a prospectively recruited intervention group exposed to ePRO-E. Covariates included technology attitudes, age, sex, education, socioeconomic status, and comorbidity. RESULTS: Intervention group ePRO intent (78.8%) was statistically significantly higher than historical control group intent (47.1%) (P = .03). Patients choosing ePRO versus paper surveys had more positive and higher technology attitudes scores (P = .03). The odds of choosing ePRO were 4.7 times higher (95% Confidence Interval [CI] = 1.2 to 17.8) (P = .02) among intervention group patients and 5.2 times higher (95% CI = 1.3 to 21.6) (P = .02) among patients with high technology attitudes scores, after controlling for covariates. However, the 80% submission rate (percentage submitting ≥80% of required surveys) in the ePRO group (30.6%) was statistically significantly lower than in the paper group (57.9%) (P = .05). CONCLUSIONS: ePRO-E exposure increased ePRO intent. High technology attitudes scores were associated with ePRO selection. Since the ePRO survey submission rate was low, additional strategies are needed to promote high-quality data submission.

Actin-binding protein profilin1 is an important determinant of cellular phosphoinositide control.

Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.

Randomized phase III trial evaluating motivational interviewing and text interventions to optimize adherence to breast cancer endocrine therapy (Alliance A191901): the GETSET protocol.

BACKGROUND: Hormone receptor-positive (HR +) breast cancer is the most common type of breast cancer in the USA but has excellent long-term outcomes in recent decades, in part due to effective oral endocrine therapy (ET). ET medications are typically prescribed for 5 to 10 years, depending on the risk of recurrence, and must be taken daily. One limiting factor to ET efficacy is nonadherence, with high-risk groups for nonadherence including younger women and Black women. METHODS: The Alliance for Clinical Trials in Oncology (Alliance) trial A191901 is an ongoing, four-arm (text message reminder (TMR), motivational interviewing (MI), TMR plus MI, or enhanced usual care) randomized clinical trial that tests the efficacy and effect of two interventions (TMR and/or MI) on improved ET adherence, patient-reported outcomes (PROs), and resource use requirements among HR + breast cancer survivors. Participants are randomized in a 1:1:1:1 ratio to the four arms. With an assumed loss to follow-up of approximately 11%, we plan to recruit 1180 participants. Randomization is stratified based on age and race to ensure balance between the arms, and we oversample younger and Black women, with each group representing 30% of the study population. Participants randomized to an intervention will actively participate in the intervention for 9 months, and all participants will be followed for adherence data and PRO endpoints, through the use of the Pillsy cap medication event monitoring system and Alliance ePRO survey app (i.e., Patient Cloud). The primary analysis will compare Pillsy-measured ET adherence among study arms at 12 months. DISCUSSION: This multisite study will not only define strategies to improve adherence to breast cancer oral therapies, but it will also potentially support strategies in large cooperative research groups that can increase delivery and tolerability of ET, involve diverse patient populations in clinical research, and engage patients effectively in interventional studies, using remote and cost-effective delivery methods. TRIAL REGISTRATION: Clinicaltrials.gov NCT04379570 . Registered on 7 May 2020.

Chemical sensors for imaging total cellular aliphatic aldehydes in live cells.

Aliphatic aldehydes are reactive electrophilic carbonyls that cross-link with DNA and proteins leading to cellular toxicity and disease pathogenesis. This toxicity is due to the cooperative effect of multiple aldehydes via a common mechanism. Therefore, live-cell imaging of total aliphatic aldehydes, small-to-long chain (C1-C10), is highly desired to decipher their physiological and pathological functions. However, sensors for imaging total cellular aliphatic aldehydes are currently lacking despite their high concentrations (∼80 to >500 µM) inside cells. Herein, we report chemical sensors that generate a benzimidazole moiety upon reaction with aliphatic aldehydes of different chain lengths (C1-C10), resulting in turn-on fluorescence. These sensors exhibit high quantum yields, high dynamic range, and enable the quantification of changes in both the exogenous administration of aldehydes and endogenous real-time formation of aliphatic aldehydes in live mammalian cells. This tool has great potential to transform aldehyde research by illuminating cellular metabolites that have remained elusive in living systems.

A novel homeostatic mechanism tunes PI(4,5)P2-dependent signaling at the plasma membrane.

The lipid molecule phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] controls all aspects of plasma membrane (PM) function in animal cells, from its selective permeability to the attachment of the cytoskeleton. Although disruption of PI(4,5)P2 is associated with a wide range of diseases, it remains unclear how cells sense and maintain PI(4,5)P2 levels to support various cell functions. Here, we show that the PIP4K family of enzymes, which synthesize PI(4,5)P2 via a minor pathway, also function as sensors of tonic PI(4,5)P2 levels. PIP4Ks are recruited to the PM by elevated PI(4,5)P2 levels, where they inhibit the major PI(4,5)P2-synthesizing PIP5Ks. Perturbation of this simple homeostatic mechanism reveals differential sensitivity of PI(4,5)P2-dependent signaling to elevated PI(4,5)P2 levels. These findings reveal that a subset of PI(4,5)P2-driven functions might drive disease associated with disrupted PI(4,5)P2 homeostasis.

Improving patient-centered communication in breast cancer: a study protocol for a multilevel intervention of a shared treatment deliberation system (SharES) within the NCI community oncology research program (NCORP) (Alliance A231901CD).

BACKGROUND: Advances in precision medicine have given oncologists new evaluative tools to better individualize treatments for patients with curable breast cancer. These innovations have revealed a need to improve patient understanding of novel, often complex information related to breast cancer treatment decisions. Ensuring patients have the emotional support to face consequential treatment decisions, as well as the opportunity to engage and discuss with their clinicians, is key to improving patient-centered communication and patient understanding. METHODS/DESIGN: This study will implement a multilevel intervention with patient and clinician components as a NCORP Cancer Care Delivery Research (CCDR) trial within the Alliance for Clinical Trials in Oncology Research Base (Alliance). The two interventions in this study, the Shared Decision Engagement System (SharES), include (1) two versions of an evidence-based patient-facing breast cancer treatment decision tool (iCanDecide +/- an emotional support module) and (2) a clinician-facing dashboard (Clinician Dashboard) that is reviewed by surgeons/clinicians and summarizes ongoing patient needs. The design is a near minimax, hybrid stepped wedge trial of SharES where both interventions are being evaluated in a crossed design over six 12-week time periods. The primary outcome (knowledge) and key secondary outcomes (i.e., self-efficacy and cancer worry) are assessed via patient report at 5 weeks after surgery. Secondary outcomes are also assessed at 5 weeks after surgery, as well as in a second survey 9 months after registration. We anticipate recruiting a total of 700 breast cancer patients (600 evaluable after attrition) from 25 surgical practices affiliated with Alliance. DISCUSSION: Upon study completion, we will have better understanding of the impact of a multilevel intervention on patient-centered communication in breast cancer with a specific focus on whether the intervention components improve knowledge and self-efficacy and reduce cancer worry. TRIAL REGISTRATION: ClinicalTrials.gov NCT04549571 . Registered on 16 September 2020.

PI(4,5)P2 diffuses freely in the plasma membrane even within high-density effector protein complexes.

The lipid phosphatidyl-D-myo-inositol-4,5-bisphosphate [PI(4,5)P2] is a master regulator of plasma membrane (PM) function. Its effector proteins regulate transport, signaling, and cytoskeletal processes that define PM structure and function. How a single type of lipid regulates so many parallel processes is unclear. We tested the hypothesis that spatially separate PI(4,5)P2 pools associate with different PM complexes. The mobility of PI(4,5)P2 was measured using biosensors by single-particle tracking. We found that PM lipids including PI(4,5)P2 diffuse rapidly (∼0.3 µm2/s) with Brownian motion, although they spend one third of their time diffusing more slowly. Surprisingly, areas of the PM occupied by PI(4,5)P2-dependent complexes did not slow PI(4,5)P2 lateral mobility. Only the spectrin and septin cytoskeletons showed reduced PI(4,5)P2 diffusion. We conclude that even structures with high densities of PI(4,5)P2 effector proteins, such as clathrin-coated pits and focal adhesions, do not corral unbound PI(4,5)P2, questioning a role for spatially segregated PI(4,5)P2 pools in organizing and regulating PM functions.

Increasing socioeconomically disadvantaged patients' engagement in breast cancer surgery decision-making through a shared decision-making intervention (A231701CD): protocol for a cluster randomised clinical trial.

INTRODUCTION: Socioeconomic disparities for breast cancer surgical care exist. Although the aetiology of the observed socioeconomic disparities is likely multifactorial, patient engagement during the surgical consult is critical. Shared decision-making may reduce health disparities by addressing barriers to patient engagement in decision-making that disproportionately impact socioeconomically disadvantaged patients. In this trial, we test the impact of a decision aid on increasing socioeconomically disadvantaged patients' engagement in breast cancer surgery decision-making. METHODS AND ANALYSIS: This multisite randomised trial is conducted through 10 surgical clinics within the National Cancer Institute Community Oncology Research Program (NCORP). We plan a stepped-wedge design with clinics randomised to the time of transition from usual care to the decision aid arm. Study participants are female patients, aged ≥18 years, with newly diagnosed stage 0-III breast cancer who are planning breast surgery. Data collection includes a baseline surgeon survey, baseline patient survey, audio-recording of the surgeon-patient consultation, a follow-up patient survey and medical record data review. Interviews and focus groups are conducted with a subset of patients, surgeons and clinic stakeholders. The effectiveness of the decision aid at increasing patient engagement (primary outcome) is evaluated using generalised linear mixed-effects models. The extent to which the effect of the decision aid intervention on patient engagement is mediated through the mitigation of barriers is tested in joint linear structural equation models. Qualitative interviews explore how barriers impact engagement, especially for socioeconomically disadvantaged women. ETHICS AND DISSEMINATION: This protocol has been approved by the National Cancer Institute Central Institutional Review Board, and Certificate of Confidentiality has been obtained. We plan to disseminate the findings through journal publications and national meetings, including the NCORP network. Our findings will advance the science of medical decision-making with the potential to reduce socioeconomic health disparities. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT03766009).

PI(4,5)P2: signaling the plasma membrane.

In the almost 70 years since the first hints of its existence, the phosphoinositide, phosphatidyl-D-myo-inositol 4,5-bisphosphate has been found to be central in the biological regulation of plasma membrane (PM) function. Here, we provide an overview of the signaling, transport and structural roles the lipid plays at the cell surface in animal cells. These include being substrate for second messenger generation, direct modulation of receptors, control of membrane traffic, regulation of ion channels and transporters, and modulation of the cytoskeleton and cell polarity. We conclude by re-evaluating PI(4,5)P2's designation as a signaling molecule, instead proposing a cofactor role, enabling PM-selective function for many proteins.

An update on genetically encoded lipid biosensors.

Specific lipid species play central roles in cell biology. Their presence or enrichment in individual membranes can control properties or direct protein localization and/or activity. Therefore, probes to detect and observe these lipids in intact cells are essential tools in the cell biologist's freezer box. Herein, we discuss genetically encoded lipid biosensors, which can be expressed as fluorescent protein fusions to track lipids in living cells. We provide a state-of-the-art list of the most widely available and reliable biosensors and highlight new probes (circa 2018-2021). Notably, we focus on advances in biosensors for phosphatidylinositol, phosphatidic acid, and PI 3-kinase lipid products.

Synthesis of L-cyclic tetrapeptides by backbone amide activation CyClick strategy.

Cyclic tetrapeptides exhibit high cellular permeability and a wide range of biological properties and thus have gained great interest in the field of medicinal chemistry. We synthesized highly strained 12-membered head to tail cyclic peptides with varying reactive amino acids, without oligomerization using the exclusively intramolecular CyClick chemistry. This occurs by a two-step process involving the low-energy formation of a 15 atom-containing cyclic imine, followed by a chemoselective ring contraction of the peptide backbone generating a highly strained 12 atom-containing cyclic tetrapeptide. This reaction exhibited high substrate scope and generated head to tail cyclic tetrapeptides with varying amino acids at the N-terminus, showing chemoselectivity without the need for side group protection.

Dosimetric comparison of volumetric modulated arc therapy (VMAT) and high-dose-rate brachytherapy (HDR-BT) for superficial skin irradiation with significant curvature in one or more planes.

PURPOSE: This study compares the plan quality of high-dose-rate brachytherapy (HDR-BT) and volumetric modulated arc therapy (VMAT) for superficial irradiation of large areas of skin with significant curvature in one or more planes. METHODS: A total of 14 patients from two centres previously treated with either HDR-BT or VMAT were retrospectively replanned using the alternative technique. Sites included scalp and lower limbs. Identical computed tomography (CT) scans, clinical target volume (CTV) and organs at risk (OARs) and prescription were used for both techniques. Conformity, skin surface dose and OAR doses were compared. RESULTS: Conformity index was consistently better with VMAT than HDR-BT (p < 0.01). Maximum skin surface dose (D0.1cc) had a higher mean of 49.6 Gy with HDR-BT compared to 31.4 Gy for VMAT (p < 0.01). Significantly smaller volumes of healthy tissue were irradiated with VMAT than with HDR-BT. This can be seen in brain volumes receiving 10, 20 and 30 Gy EQD2 and in extremities receiving 5 and 10 Gy. When close to the volume, the lens received significantly lower doses with VMAT (p < 0.01). CONCLUSION: In this small sample, VMAT gives equal coverage with lower OAR and skin surface doses than HDR-BT for both scalp and extremities. VMAT is a useful technique for treating large, superficial volumes with significant curvature in one or more planes.

Quantification of Genetically Encoded Lipid Biosensors.

Lipids, like phosphoinositides, can be visualized in living cells in real time using genetically encoded biosensors and fluorescence microscopy. Sensor localization can be quantified by determining the fluorescence intensity of each fluorophore. Enrichment of lipids at membranes can be determined by generating and applying an organelle-specific binary mask. In this chapter, we provide a detailed list of reagents and methods to visualize and quantify relative lipid levels. Applying this approach, changes in lipid levels can be assessed in cases when lipid metabolizing enzymes are mutated or otherwise altered.

Induced Dimerization Tools to Deplete Specific Phosphatidylinositol Phosphates.

Chemical dimerization systems have been used to drive acute depletion of polyphosphoinsitides (PPIns). They do so by inducing subcellular localization of enzymes that catabolize PPIns. By using this approach, all seven PPIns can be depleted in living cells and in real time. The rapid permeation of dimerizer agents and the specific expression of recruiter proteins confer great spatial and temporal resolution with minimal cell perturbation. In this chapter, we provide detailed instructions to monitor and induce depletion of PPIns in live cells.

Site-Selective Peptide Macrocyclization.

Cyclized peptides have seen a rise in popularity in the pharmaceutical industry as drug molecules. As such, new macrocyclization methodologies have become abundant in the last several decades. However, efficient methods of cyclization without the formation of side products remain a great challenge. Herein, we review cyclization approaches that focus on site-selective chemistry. Site selectivity in macrocyclization decreases the generation of side products, leading to a greater yield of the desired peptide macrocycles. We will also take an in-depth look at the new exclusively intramolecular N-terminal site-selective CyClick strategy for the synthesis of cyclic peptides. The CyClick method uses imine formation between an aldehyde and the N terminus. The imine is then trapped by a nucleophilic attack from the second amidic nitrogen in an irreversible site-selective fashion.

Probing the subcellular distribution of phosphatidylinositol reveals a surprising lack at the plasma membrane.

The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids' role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.

CyClick Chemistry for the Synthesis of Cyclic Peptides.

Here, we report a novel "CyClick" strategy for the macrocyclization of peptides that works in an exclusively intramolecular fashion thereby precluding the formation of dimers and oligomers via intermolecular reactions. The CyClick chemistry is highly chemoselective for the N-terminus of the peptide with a C-terminal aldehyde. In this protocol, the peptide conformation internally directs activation of the backbone amide bond and thereby facilitates formation of a stable 4-imidazolidinone-fused cyclic peptide with high diastereoselectivity (>99 %). This method is tolerant to a variety of peptide aldehydes and has been applied for the synthesis of 12- to 23-membered rings with varying amino acid compositions in one pot under mild reaction conditions. The reaction generated peptide macrocycles featuring a 4-imidazolidinone in their scaffolds, which acts as an endocyclic control element that promotes intramolecular hydrogen bonding and leads to macrocycles with conformationally rigid turn structures.

Genetically encoded lipid biosensors.

Lipids convey both structural and functional properties to eukaryotic membranes. Understanding the basic lipid composition and the dynamics of these important molecules, in the context of cellular membranes, can shed light on signaling, metabolism, trafficking, and even membrane identity. The development of genetically encoded lipid biosensors has allowed for the visualization of specific lipids inside individual, living cells. However, a number of caveats and considerations have emerged with the overexpression of these biosensors. In this Technical Perspective, we provide a current list of available genetically encoded lipid biosensors, together with criteria that determine their veracity. We also provide some suggestions for the optimal utilization of these biosensors when both designing experiments and interpreting results.