Streamlined Generation of CRISPR/Cas9-Mediated Single-Cell Knockout Clones in Murine Cell Lines.

Clonal cell lines harboring loss-of-function mutations in genes of interest are crucial for studying the cellular functions of the encoded proteins. Recent advances in genome engineering have converged on the CRISPR/Cas9 technology to quickly and reliably generate frameshift mutations in the target genes across various cell lines and species. Although high on-target cleavage efficiencies can be obtained reproducibly, screening and identifying clones with loss-of-function alleles remains a major bottleneck. Here, we describe a single sgRNA strategy to generate CRISPR/Cas9-mediated frameshift mutations in target genes of mammalian cell lines that can be easily and cost-effectively identified. Given the proliferation of workhorse cell lines such as N2a cells and the resulting clonal expansion of the cell type, our protocol can facilitate the isolation of knockout clonal cell lines and their genetic validation within a period of down to 6-8 weeks.

Accumulation of Non-Pathological Liver Fat Is Associated with the Loss of Glyoxalase I Activity in Humans.

The underlying molecular mechanisms for the development of non-alcoholic fatty liver (NAFL) and its progression to advanced liver diseases remain elusive. Glyoxalase 1 (Glo1) loss, leading to elevated methylglyoxal (MG) and dicarbonyl stress, has been implicated in various diseases, including obesity-related conditions. This study aimed to investigate changes in the glyoxalase system in individuals with non-pathological liver fat. Liver biopsies were obtained from 30 individuals with a narrow range of BMI (24.6-29.8 kg/m2). Whole-body insulin sensitivity was assessed using HOMA-IR. Liver biopsies were analyzed for total triglyceride content, Glo1 and Glo2 mRNA, protein expression, and activity. Liquid chromatography-tandem mass spectrometry determined liver dicarbonyl content and oxidation and glycation biomarkers. Liver Glo1 activity showed an inverse correlation with HOMA-IR and liver triglyceride content, but not BMI. Despite reduced Glo1 activity, no associations were found with elevated liver dicarbonyls or glycation markers. A sex dimorphism was observed in Glo1, with females exhibiting significantly lower liver Glo1 protein expression and activity, and higher liver MG-H1 content compared to males. This study demonstrates that increasing liver fat, even within a non-pathological range, is associated with reduced Glo1 activity.

Knock-out of dipeptidase CN2 in human proximal tubular cells disrupts dipeptide and amino acid homeostasis and para- and transcellular solute transport.

AIM: Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase-2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function. METHODS: A knockout of the CN2 gene (CNDP2-KO) was generated in human proximal tubule cells and characterized by metabolomics, RNA-seq analysis, paracellular permeability analysis and ion transport. RESULTS: CNDP2-KO in human proximal tubule cells resulted in the accumulation of cellular dipeptides, reduction of amino acids and imbalance of related metabolic pathways, and of energy supply. RNA-seq analyses indicated altered protein metabolism and ion transport. Detailed functional studies demonstrated lower CNDP2-KO cell viability and proliferation, and altered ion and macromolecule transport via trans- and paracellular pathways. Regulatory and transport protein abundance was disturbed, either as a consequence of the metabolic imbalance or the resulting functional disequilibrium. CONCLUSION: CN2 function has a major impact on intracellular amino acid and dipeptide metabolism and is essential for key metabolic and regulatory functions of proximal tubular cells. These findings deserve in vivo analysis of the relevance of CN2 for nephron function and regulation of body homeostasis.

Training the Next Generation of Data Monitoring Committee Members: An Initiative of the Heart Failure Collaboratory.

Clinical trials are vital for assessing therapeutic interventions. The associated data monitoring committees (DMCs) safeguard patient interests and enhance trial integrity, thus promoting timely, reliable evaluations of those interventions. We face an urgent need to recruit and train new DMC members. The Heart Failure Collaboratory (HFC), a multidisciplinary public-private consortium of academics, trialists, patients, industry representatives, and government agencies, is working to improve the clinical trial ecosystem. The HFC aims to improve clinical trial efficiency and quality by standardizing concepts, and to help meet the demand for experienced individuals on DMCs by creating a standardized approach to training new members. This paper discusses the HFC's training workshop, and an apprenticeship model for new DMC members. It describes opportunities and challenges DMCs face, along with common myths and best practices learned through previous experiences, with an emphasis on data confidentiality and need for quality independent statistical reporting groups.

Neuron-astrocyte metabolic coupling facilitates spinal plasticity and maintenance of inflammatory pain.

Long-lasting pain stimuli can trigger maladaptive changes in the spinal cord, reminiscent of plasticity associated with memory formation. Metabolic coupling between astrocytes and neurons has been implicated in neuronal plasticity and memory formation in the central nervous system, but neither its involvement in pathological pain nor in spinal plasticity has been tested. Here we report a form of neuroglia signalling involving spinal astrocytic glycogen dynamics triggered by persistent noxious stimulation via upregulation of the Protein Targeting to Glycogen (PTG) in spinal astrocytes. PTG drove glycogen build-up in astrocytes, and blunting glycogen accumulation and turnover by Ptg gene deletion reduced pain-related behaviours and promoted faster recovery by shortening pain maintenance in mice. Furthermore, mechanistic analyses revealed that glycogen dynamics is a critically required process for maintenance of pain by facilitating neuronal plasticity in spinal lamina 1 neurons. In summary, our study describes a previously unappreciated mechanism of astrocyte-neuron metabolic communication through glycogen breakdown in the spinal cord that fuels spinal neuron hyperexcitability.

Phenotypic and genome-wide studies on dicarbonyls: major associations to glomerular filtration rate and gamma-glutamyltransferase activity.

BACKGROUND: The dicarbonyl compounds methylglyoxal (MG), glyoxal (GO) and 3-deoxyglucosone (3-DG) have been linked to various diseases. However, disease-independent phenotypic and genotypic association studies with phenome-wide and genome-wide reach, respectively, have not been provided. METHODS: MG, GO and 3-DG were measured by LC-MS in 1304 serum samples of two populations (KORA, n = 482; BiDirect, n = 822) and assessed for associations with genome-wide SNPs (GWAS) and with phenome-wide traits. Redundancy analysis (RDA) was used to identify major independent trait associations. FINDINGS: Mutual correlations of dicarbonyls were highly significant, being stronger between MG and GO (ρ = 0.6) than between 3-DG and MG or GO (ρ = 0.4). Significant phenotypic results included associations of all dicarbonyls with sex, waist-to-hip ratio, glomerular filtration rate (GFR), gamma-glutamyltransferase (GGT), and hypertension, of MG and GO with age and C-reactive protein, of GO and 3-DG with glucose and antidiabetics, of MG with contraceptives, of GO with ferritin, and of 3-DG with smoking. RDA revealed GFR, GGT and, in case of 3-DG, glucose as major contributors to dicarbonyl variance. GWAS did not identify genome-wide significant loci. SNPs previously associated with glyoxalase activity did not reach nominal significance. When multiple testing was restricted to the lead SNPs of GWASs on the traits selected by RDA, 3-DG was found to be associated (p = 2.3 × 10-5) with rs1741177, an eQTL of NF-κB inhibitor NFKBIA. INTERPRETATION: This large-scale, population-based study has identified numerous associations, with GFR and GGT being of pivotal importance, providing unbiased perspectives on dicarbonyls beyond the current state. FUNDING: Deutsche Forschungsgemeinschaft, Helmholtz Munich, German Centre for Cardiovascular Research (DZHK), German Federal Ministry of Research and Education (BMBF).

Methods for the estimation of direct and indirect vaccination effects by combining data from individual- and cluster-randomized trials.

Both individually and cluster randomized study designs have been used for vaccine trials to assess the effects of vaccine on reducing the risk of disease or infection. The choice between individually and cluster randomized designs is often driven by the target estimand of interest (eg, direct versus total), statistical power, and, importantly, logistic feasibility. To combat emerging infectious disease threats, especially when the number of events from one single trial may not be adequate to obtain vaccine effect estimates with a desired level of precision, it may be necessary to combine information across multiple trials. In this article, we propose a model formulation to estimate the direct, indirect, total, and overall vaccine effects combining data from trials with two types of study designs: individual-randomization and cluster-randomization, based on a Cox proportional hazards model, where the hazard of infection depends on both vaccine status of the individual as well as the vaccine status of the other individuals in the same cluster. We illustrate the use of the proposed model and assess the potential efficiency gain from combining data from multiple trials, compared to using data from each individual trial alone, through two simulation studies, one of which is designed based on a cholera vaccine trial previously carried out in Matlab, Bangladesh.

Meaningful Endpoints for Idiopathic Pulmonary Fibrosis (IPF) Clinical Trials: Emphasis on 'Feels, Functions, Survives'. Report of a Collaborative Discussion in a Symposium with Direct Engagement from Representatives of Patients, Investigators, the National Institutes of Health, a Patient Advocacy Organization, and a Regulatory Agency.

Background: Idiopathic pulmonary fibrosis (IPF) carries significant mortality and unpredictable progression, with limited therapeutic options. Designing trials with patient-meaningful endpoints, enhancing the reliability and interpretability of results, and streamlining the regulatory approval process are of critical importance to advancing clinical care in IPF. Methods: A landmark in-person symposium in June 2023 assembled 43 participants from the US and internationally, including patients with IPF, investigators, and regulatory representatives, to discuss the immediate future of IPF clinical trial endpoints. Patient advocates were central to discussions, which evaluated endpoints according to regulatory standards and the FDA's 'feels, functions, survives' criteria. Results: Three themes emerged: 1) consensus on endpoints mirroring the lived experiences of patients with IPF; 2) consideration of replacing forced vital capacity (FVC) as the primary endpoint, potentially by composite endpoints that include 'feels, functions, survives' measures or FVC as components; 3) support for simplified, user-friendly patient-reported outcomes (PROs) as either components of primary composite endpoints or key secondary endpoints, supplemented by functional tests as secondary endpoints and novel biomarkers as supportive measures (FDA Guidance for Industry (Multiple Endpoints in Clinical Trials) available at: https://www.fda.gov/media/162416/download). Conclusions: This report, detailing the proceedings of this pivotal symposium, suggests a potential turning point in designing future IPF clinical trials more attuned to outcomes meaningful to patients, and documents the collective agreement across multidisciplinary stakeholders on the importance of anchoring IPF trial endpoints on real patient experiences-namely, how they feel, function, and survive. There is considerable optimism that clinical care in IPF will progress through trials focused on patient-centric insights, ultimately guiding transformative treatment strategies to enhance patients' quality of life and survival.

Impaired Detoxification of Trans, Trans-2,4-Decadienal, an Oxidation Product from Omega-6 Fatty Acids, Alters Insulin Signaling, Gluconeogenesis and Promotes Microvascular Disease.

Omega-6 fatty acids are the primary polyunsaturated fatty acids in most Western diets, while their role in diabetes remains controversial. Exposure of omega-6 fatty acids to an oxidative environment results in the generation of a highly reactive carbonyl species known as trans, trans-2,4-decadienal (tt-DDE). The timely and efficient detoxification of this metabolite, which has actions comparable to other reactive carbonyl species, such as 4-hydroxynonenal, acrolein, acetaldehyde, and methylglyoxal, is essential for disease prevention. However, the detoxification mechanism for tt-DDE remains elusive. In this study, the enzyme Aldh9a1b is identified as having a key role in the detoxification of tt-DDE. Loss of Aldh9a1b increased tt-DDE levels and resulted in an abnormal retinal vasculature and glucose intolerance in aldh9a1b-/- zebrafish. Transcriptomic and metabolomic analyses revealed that tt-DDE and aldh9a1b deficiency in larval and adult zebrafish induced insulin resistance and impaired glucose homeostasis. Moreover, alterations in hyaloid vasculature is induced by aldh9a1b knockout or by tt-DDE treatment can be rescued by the insulin receptor sensitizers metformin and rosiglitazone. Collectively, these results demonstrated that tt-DDE is the substrate of Aldh9a1b which causes microvascular damage and impaired glucose metabolism through insulin resistance.

Non-cross-linking advanced glycation end products affect prohormone processing.

Advanced glycation end products (AGEs) are non-enzymatic post-translational modifications of amino acids and are associated with diabetic complications. One proposed pathomechanism is the impaired processing of AGE-modified proteins or peptides including prohormones. Two approaches were applied to investigate whether substrate modification with AGEs affects the processing of substrates like prohormones to the active hormones. First, we employed solid-phase peptide synthesis to generate unmodified as well as AGE-modified protease substrates. Activity of proteases towards these substrates was quantified. Second, we tested the effect of AGE-modified proinsulin on the processing to insulin. Proteases showed the expected activity towards the unmodified peptide substrates containing arginine or lysine at the C-terminal cleavage site. Indeed, modification with Nε-carboxymethyllysine (CML) or methylglyoxal-hydroimidazolone 1 (MG-H1) affected all proteases tested. Cysteine cathepsins displayed a reduction in activity by ∼50% towards CML and MG-H1 modified substrates. The specific proteases trypsin, proprotein convertases subtilisin-kexins (PCSKs) type proteases, and carboxypeptidase E (CPE) were completely inactive towards modified substrates. Proinsulin incubation with methylglyoxal at physiological concentrations for 24 h resulted in the formation of MG-modified proinsulin. The formation of insulin was reduced by up to 80% in a concentration-dependent manner. Here, we demonstrate the inhibitory effect of substrate-AGE modifications on proteases. The finding that PCSKs and CPE, which are essential for prohormone processing, are inactive towards modified substrates could point to a yet unrecognized pathomechanism resulting from AGE modification relevant for the etiopathogenesis of diabetes and the development of obesity.

Estrogen-dependent expression and function of secretogranin 2a in female-specific peptidergic neurons.

Secretogranin 2 (Scg2) is a member of the secretogranin/chromogranin family of proteins that is involved in neuropeptide and hormone packaging to secretory granules and serves as a precursor for several secreted pleiotropic peptides. A recent study in zebrafish showed that the teleost Scg2 orthologs, scg2a and scg2b, play an important role in mating behavior, but its modes of action and regulatory mechanisms remain unclear. In this study, we identify scg2a in another teleost species, medaka, by transcriptomic analysis as a gene that is expressed in an ovarian secretion-dependent manner in a group of neurons relevant to female sexual receptivity, termed FeSP neurons. Investigation of scg2a expression in the FeSP neurons of estrogen receptor (Esr)-deficient medaka revealed that it is dependent on estrogen signaling through Esr2b, the major determinant of female-typical mating behavior. Generation and characterization of scg2a-deficient medaka showed no overt changes in secretory granule packaging in FeSP neurons. This, along with the observation that Scg2a and neuropeptide B, a major neuropeptide produced by FeSP neurons, colocalize in a majority of secretory granules, suggests that Scg2a mainly serves as a precursor for secreted peptides that act in conjunction with neuropeptide B. Further, scg2a showed sexually biased expression in several brain nuclei implicated in mating behavior. However, we found no significant impact of scg2a deficiency on the performance of mating behavior in either sex. Collectively, our results indicate that, although perhaps not essential for mating behavior, scg2a acts in an estrogen/Esr2b signaling-dependent manner in neurons that are relevant to female sexual receptivity.

Global Cancer Drug Development-A Report From the 2022 Accelerating Anticancer Agent Development and Validation Meeting.

Rapidly expanding systemic treatment options, combined with improved screening, diagnostic, surgical, and radiotherapy techniques, have led to improved survival outcomes for many cancers over time. However, these overall survival gains have disproportionately benefited patients in high-income countries, whereas patients in low- and middle-income countries (LMICs) continue to experience challenges in accessing timely and guideline concordant care. In September 2022, the Accelerating Anticancer Agent Development and Validation workshop was held, focusing on global cancer drug development. Panelists discussed key barriers such as the lack of diagnostic services and human resources, drug accessibility and affordability, lack of research infrastructure, and regulatory and authorization challenges, with a particular focus on Africa and Latin America. Potential opportunities to improve access and affordability were reviewed, such as the importance of prioritizing investments in diagnostics, investing health infrastructure and work force planning, coordinated drug procurement efforts and streamlined regulatory processing, incentivized pricing through regulatory change, and the importance of developing and promoting clinical trials that can answer relevant clinical questions for patients in LMICs. As a cancer community, we must continue to advocate for and work toward equitable access to high-quality interventions for patients, regardless of their geographical location.

Endothelial Notch1 signaling in white adipose tissue promotes cancer cachexia.

Cachexia is a major cause of morbidity and mortality in individuals with cancer and is characterized by weight loss due to adipose and muscle tissue wasting. Hallmarks of white adipose tissue (WAT) remodeling, which often precedes weight loss, are impaired lipid storage, inflammation and eventually fibrosis. Tissue wasting occurs in response to tumor-secreted factors. Considering that the continuous endothelium in WAT is the first line of contact with circulating factors, we postulated whether the endothelium itself may orchestrate tissue remodeling. Here, we show using human and mouse cancer models that during precachexia, tumors overactivate Notch1 signaling in distant WAT endothelium. Sustained endothelial Notch1 signaling induces a WAT wasting phenotype in male mice through excessive retinoic acid production. Pharmacological blockade of retinoic acid signaling was sufficient to inhibit WAT wasting in a mouse cancer cachexia model. This demonstrates that cancer manipulates the endothelium at distant sites to mediate WAT wasting by altering angiocrine signals.

The influence of pre-operative Computed Tomography (CT) on surgical approach and fixation for fractures of the tibial plateau.

Background: It is well known that a computed tomography (CT) scan improves the classification of tibial plateau fractures (TPF) compared with radiographs. However, it is less clear how this translates into clinical practice. The aim of this study is to establish to what extent a pre-operative CT scan alters the approach, setup and fixation choice in TPF compared to radiographs. Methods: 50 consecutive TPF with a preoperative CT and radiographic imaging available, were assessed by 4 consultant surgeons. First, anonymised radiographs were classifying according to the column classification and the planned setup, approach, and fixation technique documented. At a 1-month interval, randomised matched CT scans were assessed and the same data collected. A tibial plateau disruption score (TPDS) was derived for all 4 quadrants (no injury = 0, split = 1, split/depression = 2 and depression = 3). Radiograph and CT TPDS were assessed using an unpaired T-test. Results: 26 female and 24 male patients, mean age 50.3, were included. Mean TPDS on radiographs and CT scans were 2.77 and 3.17 respectively. A significantly higher CT TPDS, of 0.4 (95%CI 0.10-0.71)[P = 0.0093] was observed, demonstrating that radiographs underestimate the extent of injury. The surgical approach changed in 28.5% of cases, thus influencing a change in the patient setup in theatre in 27% of cases. Identification of fractures within a column changed in 34% of cases. A high intra-observer reliability was observed when surgeons were asked to repeat their assessment in a third round at a further one month interval. Conclusion: A pre-operative CT scan has a significant effect on the approach required to fix TPF. This therefore influences the setup of the patient and can justifiably be requested as part of pre-operative planning.

The effect of GLP-1 receptor agonist lixisenatide on experimental diabetic retinopathy.

AIMS: Glucagon-like peptide-1 receptor agonists are effective treatments for type 2 diabetes, effectively lowering glucose without weight gain and with low risk for hypoglycemia. However, their influence on the retinal neurovascular unit remains unclear. In this study, we analyzed the effects of the GLP-1 RA lixisenatide on diabetic retinopathy. METHODS: Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated C. elegans, respectively. In STZ-diabetic Wistar rats, acellular capillaries and pericytes (quantitative retinal morphometry), neuroretinal function (mfERG), macroglia (GFAP western blot) and microglia (immunohistochemistry) quantification, methylglyoxal (LC-MS/MS) and retinal gene expressions (RNA-sequencing) were determined. The antioxidant properties of lixisenatide were tested in C. elegans. RESULTS: Lixisenatide had no effect on glucose metabolism. Lixisenatide preserved the retinal vasculature and neuroretinal function. The macro- and microglial activation was mitigated. Lixisenatide normalized some gene expression changes in diabetic animals to control levels. Ets2 was identified as a regulator of inflammatory genes. In C. elegans, lixisenatide showed the antioxidative property. CONCLUSIONS: Our data suggest that lixisenatide has a protective effect on the diabetic retina, most likely due to a combination of neuroprotective, anti-inflammatory and antioxidative effects of lixisenatide on the neurovascular unit.

Glyoxal in hyperglycaemic ischemic stroke - a cohort study.

BACKGROUND: Hyperglycaemia is frequent in acute ischemic stroke and denotes a bad prognosis, even in the absence of pre-existing diabetes. However, in clinical trials treatment of elevated glucose levels with insulin did not improve stroke outcome, suggesting that collateral effects rather than hyperglycaemia itself aggravate ischemic brain damage. As reactive glucose metabolites, glyoxal and methylglyoxal are candidates for mediating the deleterious effects of hyperglycaemia in acute stroke. METHODS: In 135 patients with acute stroke, we used liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to measure glyoxal, methylglyoxal and several of their glycated amino acid derivatives in serum. Results were verified in a second cohort of 61 stroke patients. The association of serum concentrations with standard stroke outcome scales (NIHSS, mRS) was tested. RESULTS: Glucose, glyoxal, methylglyoxal, and the glyoxal-derived glycated amino acid Nδ-(5-hydro-4-imidazolon-2-yl)ornithine (G-H1) were positively correlated with a bad stroke outcome at 3 months as measured by mRS90, at least in one of the two cohorts. However, the glycated amino acids Nε-carboxyethyllysine (CEL) and in one cohort pyrraline showed an inverse correlation with stroke outcome probably reflecting lower food intake in severe stroke. Patients with a poor outcome had higher serum concentrations of glyoxal and methylglyoxal. CONCLUSIONS: The glucose-derived α-dicarbonyl glyoxal and glycated amino acids arising from a reaction with glyoxal are associated with a poor outcome in ischemic stroke. Thus, lowering α-dicarbonyls or counteracting their action could be a therapeutic strategy for hyperglycaemic stroke.

Bringing data monitoring committee charters into the sunlight.

Clinical trials investigating novel or high risk interventions, or studying vulnerable participants, often use a data monitoring committee to oversee the progress of the trial. The data monitoring committee serves both an ethical and a scientific function, by protecting the interests of trial participants while ensuring the integrity of the trial results. A data monitoring committee charter, which typically describes the procedures by which data monitoring committees operate, contains details about the data monitoring committee's organizational structure, membership, meeting frequency, sequential monitoring guidelines, and the overall contents of data monitoring committee reports for interim review. These charters, however, are generally not reviewed by outside entities and are rarely publicly available. The result is that a key component of trial oversight remains in the dark. We recommend that ClinicalTrials.gov modify its system to allow uploading of data monitoring committee charters, as is already possible for other important study documents and that clinical trialists take advantage of this opportunity to voluntarily upload the data monitoring committee charter for trials that have one. The resulting cache of publicly available data monitoring committee charters should provide important insights for those interested in a particular trial, as well as for meta-researchers who wish to understand and potentially improve how this important component of trial oversight is actually being applied.

The Data Monitoring Committee: A Collective or a Collection?

In this commentary, we urge that a Data Monitoring Committee (DMC) should operate as a collective, that is, as a unitary whole. In so doing, its recommendations should emerge through a consensus development process, not through a vote of the members. The summary notes of its closed session, that is, its minutes, should report the recommendations of the DMC and, if necessary, the justification for those recommendations; it should not attribute opinions to individual members. Importantly, the proceedings of the DMC meetings should not be electronically recorded.

Finding the (biomarker-defined) subgroup of patients who benefit from a novel therapy: No time for a game of hide and seek.

An important element of precision medicine is the ability to identify, for a specific therapy, those patients for whom benefits of that therapy meaningfully exceed the risks. To achieve this goal, treatment effect usually is examined across subgroups defined by a variety of factors, including demographic, clinical, or pathologic characteristics or by molecular attributes of patients or their disease. Frequently such subgroups are defined by the measurement of biomarkers. Even though such examination is necessary when pursuing this goal, the evaluation of treatment effect across a variety of subgroups is statistically fraught due to both the danger of inflated false-positive error rate from multiple testing and the inherent insensitivity to how treatment effects differ across subgroups.Pre-specification of subgroup analyses with appropriate control of false-positive (i.e. type I) error is recommended when possible. However, when subgroups are specified by biomarkers, which could be measured by different assays and might lack established interpretation criteria, such as cut-offs, it might not be possible to fully specify those subgroups at the time a new therapy is ready for definitive evaluation in a Phase 3 trial. In these situations, further refinement and evaluation of treatment effect in biomarker-defined subgroups might have to take place within the trial. A common scenario is that evidence suggests that treatment effect is a monotone function of a biomarker value, but optimal cut-offs for therapy decisions are not known. In this setting, hierarchical testing strategies are widely used, where testing is first conducted in a particular biomarker-positive subgroup and then is conducted in the expanded pool of biomarker-positive and biomarker-negative patients, with control for multiple testing. A serious limitation of this approach is the logical inconsistency of excluding the biomarker-negatives when evaluating effects in the biomarker-positives, yet allowing the biomarker-positives to drive the assessment of whether a conclusion of benefit could be extrapolated to the biomarker-negative subgroup.Examples from oncology and cardiology are described to illustrate the challenges and pitfalls. Recommendations are provided for statistically valid and logically consistent subgroup testing in these scenarios as alternatives to reliance on hierarchical testing alone, and approaches for exploratory assessment of continuous biomarkers as treatment effect modifiers are discussed.