Moving targets of neurotoxins: Proteases that cleave protein targets at specific sequences control many biological functions. The ability to reprogram proteases to cleave new sequences of our choosing would enable new therapeutic and biotechnological applications. Blum et al. report a laboratory evolution method to rapidly evolve proteases that cut new protein sequences and lose their ability to cut nontarget sequences (see the Perspective by Stenmark). Using this method, they evolved botulinum neurotoxin proteases, an important class of enzymes used in patients, to selectively cleave new targets, including a protein unrelated to those natively cleaved by these proteases. This work establishes a powerful approach to generate proteases with tailor-made specificities. Science , this issue p. 803 ; see also p. 782

Neuroblastoma (NB) is the most common extracranial solid tumor in children and is characterized by marked clinical heterogeneity and poor prognosis. MYCN amplification drives NB tumorigenesis through epigenetic reprogramming and is frequently accompanied by a copy-number gain of the long arm of chromosome 17 (17q). Epigenetic dysregulation of enhancer landscapes-particularly large regulatory elements termed super‑enhancers (SEs), which are enriched for H3K27ac and bound by lineage-specific master transcription factors (TFs)-establishes distinct NB cellular identities and states. These SE domains demarcate oncogenes that function as critical regulators of cell proliferation and apoptosis. Therefore, SE-driven genes represent tumor vulnerabilities, offering selective therapeutic opportunities. By integrating ATAC-seq data from 22 NB cell lines, the intratumoral heterogeneity of MYCN-amplified NB was characterized at the level of chromatin accessibility. Subsequently, based on H3K27ac ChIP-seq data from 38 NB cell lines, the ROSE algorithm was employed to identify SE-driven oncogenes. The synergistic mechanism between MYCN amplification and the 17q SE-driven gene MSI2 was investigated through genome‑wide CRISPR/Cas9 loss‑of‑function screens. At single‑cell resolution, we conducted a comprehensive analysis of the characteristics of heterogeneous tumor subpopulations and their immune microenvironment features. This analysis was performed using multiple bioinformatics workflows, including AUCell scoring, SCENIC analysis, copy‑number inference, cell differentiation‑state evaluation, neighborhood abundance tests, and separability tests. Finally, functional validation was performed using NB cell lines (MYCN‑amplified and non‑amplified) to assess gene perturbations. Pronounced epigenetic heterogeneity was observed within MYCN-amplified NB. MSI2 is an SE‑driven oncogene and is highly expressed in MYCN‑amplified NB. MSI2 and MYCN are co-expressed, may be mutually dependent, and are both correlated with cell cycle-related pathways. At the single-cell level, we identified and redefined an NB-MSI2 + MYCN+ subtype characterized by malignant transcriptional features, an immunosuppressive microenvironment, and poor patient prognosis. Building on this, combined targeting of MSI2 and MYCN markedly reduced proliferation and migration in MYCN‑amplified NB cells. The NB‑MSI2 + MYCN+ subtype defines a clinically aggressive, therapy‑refractory state characterized by high proliferation, metabolic reprogramming, and immunosuppression. For patients with MYCN-amplified NB, MSI2 is both a prognostic biomarker and a candidate therapeutic target.

Recurrent urinary tract infections (UTIs) caused by multidrug-resistant uropathogenic Escherichia coli (UPEC) demand novel antimicrobial approaches. This study evaluated the membrane-intercalating conjugated oligoelectrolyte COE-D8 against 93 clinical UPEC isolates. While >75% of isolates exhibited high-level resistance to first-line antibiotics (MIC > 512 μg/mL), 94.6% remained susceptible to COE-D8 at ≤32 μg/mL. COE-D8 eliminated bacteria fourfold faster than standard antibiotics through membrane disruption driven by a dimensional mismatch with the lipid bilayer. To mitigate the inherent cytotoxicity of COE-D8 alone, we developed a synergistic co-administration strategy with fosfomycin. This approach achieved a significant dose-sparing effect, rescuing 7-day murine survival from 40% to 80% in safety models without compromising efficacy. Furthermore, resistance induction studies identified a high barrier to adaptation, with survival primarily mediated by mzrA mutations. These findings establish the COE-D8/fosfomycin combination as a host-friendly, mechanism-driven strategy against refractory UPEC.

Recurrent urinary tract infections (UTIs) are increasingly associated with antibiotic resistance, posing significant clinical challenges. This study evaluates the antimicrobial efficacy of the membrane-intercalating conjugated oligoelectrolyte COE-D8 against uropathogenic Escherichia coli (UPEC). Among 93 clinical UPEC isolates, over 50% demonstrated resistance to first-line antibiotics, with compound sulfamethoxazole showing the highest resistance (75% at a minimum inhibitory concentration (MIC) >512 μg/mL). COE-D8 significantly reduced resistance rates, achieving less than 50% at 32 μg/mL and below 5% at 512 μg/mL, while demonstrating at least fourfold faster bacterial elimination than standard antibiotics. Mechanistic studies revealed that COE-D8 disrupts bacterial membranes, inducing rapid lysis. However, its cytotoxicity in mammalian cells at 2 μg/mL limits clinical application. To address this, a combination therapy with fosfomycin was explored, significantly reducing COE-D8 toxicity by at least fourfold in vitro. In mouse models, fosfomycin co-administration mitigated COE-D8-induced toxicity and prevented mortality at higher doses without compromising antimicrobial efficacy. These findings highlight the potential of COE-D8, particularly in combination with fosfomycin, as a promising strategy to combat antibiotic-resistant UPEC while addressing toxicity challenges.