A single-chain fab derived drug conjugate for HER2 specific delivery.

Despite the development of antibody-drug conjugates, the fragment Fab-based drug conjugates offer some unique capabilities in terms of safety, clearance, penetration and others. Current methods for preparing Fab drug conjugates are limited by the availability and stability of Fab proteins, leaving reports on this rare. Here, we found that a single-chain scaffold of Fab enables stabilization of the paired structure and supports high-yield expression in bacteria cytoplasm. Furthermore, we conjugated anti-neoplastic agent SN38 to the C-terminus by sortase A ligation and generated a homogenous Fab conjugate with the drug-to-Fab ratio of 1. The resulting anti-HER2 Fab-SN38 conjugate demonstrated potent and antigen-dependent cell-killing ability with the aid of its special cathepsin-triggered cyclization-promoted release mechanism. In vivo, Fab-SN38 can prevent growths of HER2-positive tumors in athymic mice and be well tolerated to the treatment at 7 mg/kg per dose. Anti-tumor activity, high dose tolerance and penetration advantage observed in this study would merit Fab conjugate investigation in target chemotherapy.

Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

Review of Atrioventricular Node Ablation Combined with Permanent His-Purkinje Conduction System Pacing in Patients with Atrial Fibrillation with Heart Failure.

With the advancement of pacing technologies, His-Purkinje conduction system pacing (HPCSP) has been increasingly recognized as superior to conventional right ventricular pacing (RVP) and biventricular pacing (BVP). This method is characterized by a series of strategies that either strengthen the native cardiac conduction system or fully preserve physical atrioventricular activation, ensuring optimal clinical outcomes. Treatment with HPCSP is divided into two pacing categories, His bundle pacing (HBP) and left bundle branch pacing (LBBP), and when combined with atrioventricular node ablation (AVNA), can significantly improve left ventricular (LV) function. It effectively prevents tachycardia and regulates ventricular rates, demonstrating its efficacy and safety across different QRS wave complex durations. Therefore, HPCSP combined with AVNA can alleviate symptoms and improve the quality of life in patients with persistent atrial fibrillation (AF) who are unresponsive to multiple radiofrequency ablation, particularly those with concomitant heart failure (HF) who are at risk of further deterioration. As a result, this "pace and ablate" strategy could become a first-line treatment for refractory AF. As a pacing modality, HBP faces challenges in achieving precise localization and tends to increase the pacing threshold. Thus, LBBP has emerged as a novel approach within HPCSP, offering lower thresholds, higher sensing amplitudes, and improved success rates, potentially making it a preferable alternative to HBP. Future large-scale, prospective, and randomized controlled studies are needed to evaluate patient selection and implantation technology, aiming to clarify the differential clinical outcomes between pacing modalities.

Cardiac resynchronization therapy guided by interventricular conduction delay: How to choose between biventricular pacing or conduction system pacing.

BACKGROUND: Biventricular pacing (BIV) is the gold standard for cardiac resynchronization therapy (CRT). Thirty percent of patients do not respond to CRT. Conduction system pacing (CSP) represents a viable alternative. Interventricular conduction delay (IVCD), as electrical desynchrony marker, is a CRT response predictor. The aim of this study was to determine the incidence of CRT responders by selecting the best approach between BIV and CPS based on intraoperative IVCD measurement in patients with HFrEF and LBBB. METHODS: Ninety-six patients were randomly assigned in a 1:1 ratio to either a standard BIV group(control group, CG) or a group where the CRT approach was determined based on IVCD evaluation(study group, SG). If the right ventricular sensed electrogram (RVs)-left ventricular sensed electrogram (LVs) interval was ≥100 ms, the lead was left in its original position; otherwise, the LV lead was removed, and CSP was performed instead. Clinical, EKG, and echocardiographic features have been assessed pre- and 6 months post-implant. Echocardiographic and clinical responder were evaluated. RESULTS: Thirty-seven percent of patients in the SG underwent CSP, as the operative algorithm. The incidence of CRT responders was significantly higher in the SG (echocardiographic criterion: 92.5% vs. 69.8%, p:.009; clinical criterion 87.5% vs. 62.8%, p:.014). The SG showed a significantly greater difference in EF between pre- and post-implant as well as reduced end-diastolic and systolic volumes. Univariate and multivariate regression analysis indicated that enrollment in the SG was the only factor associated with CRT response. CONCLUSION: Intraoperative assessment of IVCD could help determine the optimal CRT approach between BIV and CSP, leading to a significant improvement in the rate of CRT responders.

Right ventriculography to guide left bundle branch pacing in pacing-induced cardiomyopathy: a novel case report.

Background: There is emerging evidence for the potential utility of left bundle branch area pacing (LBBAP), as an alternative to conventional cardiac resynchronization therapy (CRT). The utility of right ventriculography by way of power injector to facilitate lead placement has not yet been reported in the literature. Case summary: A 79-year-old female, with a background of poorly rate-controlled atrial fibrillation, presented with worsening dyspnoea. She had recently undergone single-chamber pacemaker insertion prior to an atrioventricular nodal (AVN) ablation, owing to failure in achieving successful CRT coronary sinus lead placement. She had clinical evidence of volume overload, and her electrocardiogram demonstrated right ventricular pacing. Echocardiography demonstrated left ventricular (LV) impairment, with an ejection fraction (EF) of 35%, and severe functional mitral regurgitation (MR). Her diagnosis was overall consistent with pacing-induced cardiomyopathy (PIC). In this patient, the use of right ventriculography, using power-injector-delivered contrast, successfully facilitated placement of an LBBAP lead, with confirmation of good threshold and sensing parameters. Following an upgrade to conduction system pacing, the patient recovered well. On recent follow-up, repeat echocardiography (24 months post initial presentation) demonstrated improved LV function (EF 45% from 35%) and only mild-to-moderate MR. Discussion: In conclusion, we demonstrate the utility of right ventriculography to facilitate placement of an LBBAP lead, successfully treating a patient who developed PIC from chronic right ventricular pacing following AVN ablation.

FCGR2/3 polymorphisms are associated with susceptibility to Kawasaki disease but do not predict intravenous immunoglobulin resistance and coronary artery aneurysms.

Introduction: Kawasaki disease (KD) is a pediatric vasculitis that can result in coronary artery aneurysm (CAA) formation, which is a dangerous complication. Treatment with intravenous immunoglobulin (IVIg) significantly decreases the risk of CAA, possibly through competitive binding to Fc-gamma receptors (FcγRs), which reduces the binding of pathological immune complexes. However, ~20% of children have recrudescence of fever and have an increased risk of CAA. Therefore, we aimed to identify genetic markers at the FCGR2/3 locus associated with susceptibility to KD, IVIg resistance, or CAA. Materials and methods: We investigated the association of single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs) at the FCGR2/3 locus with KD susceptibility, IVIg resistance, and CAA risk using a family-based test (KD susceptibility) and case-control analyses (IVIg resistance and CAA risk) in different cohorts, adding up to a total of 1,167 KD cases. We performed a meta-analysis on IVIg resistance and CAA risk including all cohorts supplemented by previous studies identified through a systematic search. Results: FCGR2A-p.166His was confirmed to be strongly associated with KD susceptibility (Z = 3.17, p = 0.0015). In case-control analyses, all of the investigated genetic variations at the FCGR2/3 locus were generally not associated with IVIg resistance or with CAA risk, apart from a possible association in a Polish cohort for the FCGR3B-NA2 haplotype (OR = 2.15, 95% CI = 1.15-4.01, p = 0.02). Meta-analyses of all available cohorts revealed no significant associations of the FCGR2/3 locus with IVIg resistance or CAA risk. Discussion: FCGR2/3 polymorphisms are associated with susceptibility to KD but not with IVIg resistance and CAA formation. Currently known genetic variations at the FCGR2/3 locus are not useful in prediction models for IVIg resistance or CAA risk.

A New Dimension in Cardiac Imaging: 3D Exploration of the Atrioventricular Conduction Axis with Hierarchical Phase-contrast Tomography.

Much of our understanding of the atrioventricular conduction axis has been derived from early 20th-century histological investigations. These studies, while foundational, are constrained by their two-dimensional representation of complex, three-dimensional anatomy. The variability in the course of the atrioventricular conduction axis, and its relationship to surrounding cardiac structures, necessitates a more advanced imaging approach. Utilizing hierarchical phase-contrast tomography (HiP-CT) of an autopsied heart specimen with cellular resolution, this review provides a contemporary understanding of the atrioventricular conduction axis. By correlating these findings with three-dimensional computed tomographic reconstructions in living patients, we offer clinicians the insights needed accurately to predict the location of the atrioventricular conduction axis. This novel approach overcomes the inherent limitations of two-dimensional histology, enhancing our ability to understand and visualize the intricate relationships of the conduction axis within the heart.

Stylet-driven Leads or Lumenless Leads for Conduction System Pacing.

Stylet-driven leads have been recently introduced for conduction system pacing, while most of the previous experience has been obtained with lumenless leads. Design and structural characteristics of both lead types are significantly different, resulting in different implant techniques and independent learning curves. Lead performance appears to be comparable, whereas data on direct comparison of clinical outcomes are scarce. Currently, there are no specific clinical scenarios favouring the use of one lead type over another and the decision should rely on the individual experience of the implanter.

Evolution and development of the conduction system in the vertebrate heart: a role for hemodynamics and the epicardium.

Development of the heart is a very intricate and multiplex process as it involves not only the three spatial dimensions but also the fourth or time dimension. Over time, the heart of an embryo needs to adapt its function to serve the increasing complexity of differentiation and growth towards adulthood. It becomes even more perplexing by expanding time into millions of years, allocating related species in the tree of life. As the evolution of soft tissues can hardly be studied, we have to rely on comparative embryology, supported heavily by genetic and molecular approaches. These techniques provide insight into relationships, not only between species, but also between cell populations, signaling mechanisms, molecular interactions and physical factors such as hemodynamics. Heart development depends on differentiation of a mesodermal cell population that - in more derived taxa - continues in segmentation of the first and second heart field. These fields deliver not only the cardiomyocytes, forming the three-dimensionally looping cardiac tube as a basis for the chambered heart, but also the enveloping epicardium. The synchronized beating of the heart is then organized by the conduction system. In this Review, the epicardium is introduced as an important player in cardiac differentiation, including the conduction system.

Novel cellular functions of Cys2-His2 zinc finger proteins in anthracnose development and dissemination on pepper fruits by Colletotrichum scovillei.

Colletotrichum species are notorious for causing anthracnose on many fruits, leading to significant economic losses worldwide. As a model, we functionally characterized cys2-his2 (C2H2) zinc finger proteins (CsCZFs) in Colletotrichum scovillei, a major causal agent of pepper fruit anthracnose in many countries. In all, 62 CsCZFs were identified by in silico genomic analysis. Twelve were selected based on their expression profiles to generate targeted deletion mutants for functional investigation. ΔCsczf1 markedly reduced conidiation and constitutive expression of CsCZF1 partially recovered conidiation in an asexual reproduction-defective mutant, ΔCshox2. Deletion of CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, impaired autophagy in C. scovillei. ΔCsczf9 was defective in surface recognition, appressorium formation, and suppression of host defenses. CsCZF9 was identified as an essential and novel regulator under the control of the mitogen-activated protein kinase (CsPMK1) in an early step of appressorium development in C. scovillei. This study provides novel insights into CsCZF-mediated regulation of differentiation and pathogenicity in C. scovillei, contributing to understanding the regulatory mechanisms governing fruit anthracnose epidemics.IMPORTANCEThe phytopathogenic fungus Colletotrichum scovillei is known to cause serious anthracnose on chili pepper. However, the molecular mechanism underlying anthracnose caused by this fungus remains largely unknown. Here, we systematically analyzed the functional roles of cys2-his2 zinc finger proteins (CsCZFs) in the dissemination and pathogenic development of this fungus. Our results showed that CsCZF1 plays an important role in conidiation and constitutive expression of CsCZF1 restored conidiation in an asexual reproduction-defective mutant, ΔCshox2. The CsCZF9, a novel target of the mitogen-activated protein kinase (CsPMK1), is essential for surface recognition to allow appressorium formation and suppression of host defenses in C. scovillei. The CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, is involved in the autophagy of C. scovillei. Our findings reveal a comprehensive mechanism underlying CsCZF-mediated regulation of differentiation and pathogenicity of C. scovillei, which contributes to the understanding of fruit anthracnose epidemics and the development of novel strategies for disease management.

His bundle pacing combined with atrioventricular node ablation for atrial fibrillation: a systematic review and meta-analysis.

INTRODUCTION AND OBJECTIVE: His bundle pacing (HBP) could replace failed biventricular pacing (BVP) in guidelines (IIa Indication), but the high capture thresholds and backup lead pacing requirements limit its development. We assessed the efficacy and safety of HBP combined with atrioventricular node ablation (AVNA) for atrial fibrillation (AF) and compared with BVP and left bundle branch pacing (LBBP). METHODS: We reviewed PubMed, Embase, Web of Science, and Cochrane Library databases on left ventricular ejection fraction (LVEF), New York Heart Association (NYHA) score, QRS duration (QRSd), and pacing threshold. RESULTS: Thirteen studies included 1115 patients (639 with HBP, 338 with BVP, and 221 with LBBP). Compared with baseline, HBP improved LVEF (mean difference [MD]: 9.24 [6.10, 12.37]; p < 0.01), reduced NYHA score (MD: -1.12 [-1.34, -0.91]; p < 0.01), increased QRSd (MD: 10.08 [4.45, 15.70]; p < 0.01), and rose pacing threshold (MD: 0.16 [0.05, 0.26]; p < 0.01). HBP had comparable efficacy to BVP and LBBP and lower QRSd (p < 0.05). HBP had a lower success rate (85.97%) and more complications (16.1%). CONCLUSION: HBP combined with AVNA is effective for AF, despite having a lower success rate and more complications. Further trials are required to determine whether HBP is superior to BVP and LBBP.

His-bundle pacing from the right atrium: Solving pacemaker implantation challenges post-TriClip.

INTRODUCTION: This case report highlights the novel role of His-bundle pacing (HBP) from right atrium, not just for preserving cardiac function, but also for avoiding interference with TriClip devices. METHODS AND RESULTS: A 78-year-old female with severe tricuspid regurgitation received two TriClip devices. Postprocedure, frequent significant sinus pauses required a pacemaker. HBP was chosen to avoid lead complications. Under local anesthesia, a His pacing lead was inserted via the axillary vein using specialized catheter. Follow-ups over 2.5 years showed stable parameters with no complications. CONCLUSION: HBP is effective for patients with TriClip devices, ensuring optimal cardiac function and lead stability.

Decremental properties of a concealed nodoventricular pathway.

INTRODUCTION: The decremental properties of the nodoventricular pathway (NVP) are uncertain. METHODS AND RESULTS: During short RP supraventricular tachycardia, a His-refractory premature ventricular contraction (PVC) consistently terminated the tachycardia without atrial capture immediately after the PVC. Whereas a slightly earlier PVC failed to reset the subsequent His but terminated the tachycardia without atrial capture one cycle later. CONCLUSION: These observations are diagnostic of slow-fast atrioventricular nodal reentrant tachycardia (AVNRT) with a bystander concealed-NVP and can be explained by decremental properties in the NVP itself; greater prematurity of the PVC resulted in more decremental conduction over the NVP, causing the AVNRT termination one cycle later.

Variety is the spice of life: nongenetic variation in life histories influences population growth and evolvability.

Individual vital rates are key determinants of lifetime reproductive success, and variability in these rates shapes population dynamics. Previous studies have found that this vital rate hetero- geneity can influence demographic properties including population growth rates, however, the explicit effects of the amount of variation within and the covariance between vital rates that can also vary throughout the lifespan on population growth remains unknown. Here, we explore the analytical consequences of nongenetic heterogeneity on long-term population growth rates and rates of evolution by modifying traditional age-structured population projection matrices to incorporate variation among individual vital rates. The model allows vital rates to be permanent throughout life ("fixed condition") or to change over the lifespan ("dynamic condition"). We reduce the complexity associated with adding individual heterogeneity to age-structured models through a novel application of matrix collapsing ("phenotypic collapsing"), showing how to col- lapse in a manner that preserves the asymptotic and transient dynamics of the original matrix. The main conclusion is that nongenetic individual heterogeneity can strongly impact the long-term growth rate and rates of evolution. The magnitude and sign of this impact depends heavily on how the heterogeneity covaries across the lifespan of an organism. Our results emphasize that nongenetic variation cannot simply be viewed as random noise, but rather that it has consistent, predictable effects on fitness and evolvability.

Left ventricular volumes and function in successful and failed His-BundLe Pacing. A comparative prospective study.

INTRODUCTION: Initial data suggest that His Bundle Pacing (HBP) could preserve long-term cardiac structure and function better than Right Ventricular Pacing (RVP), but evidence is limited. METHODS: We studied consecutive patients with baseline ejection fraction (EF) ≥ 50% who underwent HBP attempt, either successful (HBP group) or failed (RVP group). Two-dimensional (2D) and three-dimensional (3D) echocardiography were carried out at baseline and after 6 months of ventricular pacing burden > 20%. RESULTS: Among 68 patients, 40 underwent successful HBP, and 28 RVP. The HBP and RVP groups did not differ for age, sex and pacing indication. At baseline, the HBP and RVP groups did not differ for 2D EF (62% vs. 62%), 3D EF (60% vs. 63%), 2D (-19% vs. -19%) and 3D global longitudinal strain (GLS) (-15% vs. -16%). After 6 months, 2D EF (-3.86%) and 3D EF (-5.71%) significantly decreased in the RVP group and did not change in the HBP group (p for interaction .006 and <.001, respectively). 2D GLS (3.08%) and 3D GLS (2.22%) significantly increased in the RVP group, but did not change in the HBP group (p for interaction .013 and <.016, respectively). Pacing induced cardiomyopathy (PICM) (EF drop ≥ 10% and EF < 50%) occurred in 14% (RVP) versus 0% (HBP) of patients (p = .025). CONCLUSIONS: Successful HBP was superior to RVP in preserving LV systolic function despite a high ventricular pacing burden, and was less frequently associated with PICM.

Anatomical Ablation of the Atrioventricular Node.

Background: Atrioventricular (AV) conduction ablation has been achieved by targeting the area of penetration of the conduction axis as defined by recording a His bundle potential. Ablation of the His bundle may reduce the possibility of a robust junctional escape rhythm. It was hypothesised that specific AV nodal ablation is feasible and safe. Methods: The anatomical position of the AV node in relation to the site of penetration of the conduction axis was identified as described in dissections and histological sections of human hearts. Radiofrequency (RF) ablation was accomplished based on the anatomical criteria. Results: Specific anatomical ablation of the AV node was attempted in 72 patients. Successful AV nodal ablation was accomplished in 63 patients (87.5%), following 60 minutes (IQR 50-70 minutes) of procedure time, 3.4 minutes (IQR 2.4-5.5 minutes) of fluoroscopy time, and delivery of 4 (IQR 3-6) RF lesions. An escape rhythm was present in 45 patients (71%), and the QRS complex was similar to that before ablation in all 45 patients. Atropine was administered in six patients after the 10-min waiting period and did not result in restoration of conduction. In nine patients, AV conduction could not be interrupted, and AV block was achieved with ablation of the His after delivery of 12 (IQR 8-15) RF lesions. No cases of sudden death were encountered, and all patients had persistent AV block during a median 10.5 months (IQR 5-14 months) of follow-up. Conclusion: Anatomical ablation of the AV node is feasible and safe, and results in an escape rhythm similar to that before ablation.

A unified purification method for actin-binding proteins using a TEV-cleavable His-Strep-tag.

The actin cytoskeleton governs the dynamic functions of cells, ranging from motility to phagocytosis and cell division. To elucidate the molecular mechanism, in vitro reconstructions of the actin cytoskeleton and its force generation process have played essential roles, highlighting the importance of efficient purification methods for actin-binding proteins. In this study, we introduce a unified purification method for actin-binding proteins, including capping protein (CP), cofilin, ADF, profilin, fascin, and VASP, key regulators in force generation of the actin cytoskeleton. Exploiting a His-Strep-tag combined with a TEV protease cleavage site, we purified these diverse actin-binding proteins through a simple two-column purification process: initial purification through a Strep-Tactin column and subsequent tag removal through the reverse purification by a Ni-NTA column. Biochemical and microscopic assays validated the functionality of the purified proteins, demonstrating the versatility of the approach. Our methods not only delineate critical steps for the efficient preparation of actin-binding proteins but also hold the potential to advance investigations of mutants, isoforms, various source species, and engineered proteins involved in actin cytoskeletal dynamics.•Unified purification method for various actin-binding proteins.•His-Strep-tag and TEV protease cleavage for efficient purification.•Functional validation through biochemical and microscopic assays.

Facile Synthesis of Aptamer-Functionalized Polydopamine-Coated Magnetic Graphene Oxide Nanocomposites for Highly Efficient Purification of His-Tagged Proteins.

Recombinant proteins hold significant importance in numerous disciplines. As the demand for expressing and purifying these proteins grows, the scientific community is in dire need of a simple yet versatile methodology that can efficiently purify these proteins. Aptamers as synthetic nucleic acid-based ligands with high affinity have shown promise in this regard, as they can capture targets through molecular recognition. In this study, novel aptamer-functionalized polydopamine-coated magnetic graphene oxide nanocomposites were facilely prepared, achieving an impressive average aptamer coverage density (45 nmol/mg). These nanocomposites exhibited a uniform structure and robust magnetic responsiveness. The findings indicated that they possess several advantages, such as rapid adsorption, substantial capacity (171.4 mg/g), and excellent reusability. Notably, due to the inherent properties of nucleic acids, the immobilized aptamer-magnetic beads can be utilized repeatedly with high purification efficiency. Finally, the nanocomposites were further employed to purify His-tagged proteins from actual samples. Remarkably, they were able to selectively and efficiently isolate His-tagged retinoid X receptor alpha protein from complex Escherichia coli lysate. The purified His-tagged retinoid X receptor alpha protein was analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This confirmed the efficacy of developed nanocomposites, reinforcing their vast potential for purification of His-tagged recombinant proteins.

1H, 15N and 13C resonance assignments of the S2A and H64A double mutant of human carbonic anhydrase II.

Protein-water interactions profoundly influence protein structure and dynamics. Consequently, the function of many biomacromolecules is directly related to the presence and exchange of water molecules. While structural water molecules can be readily identified through X-ray crystallography, the dynamics within functional protein-water networks remain largely elusive. Therefore, to understand the role of biological water in protein dynamics and function, we have introduced S2A and H64A mutations in human Carbonic Anhydrase II (hCAII), a model system to study protein-water interactions. The mutations of serine to alanine at position 2 and histidine to alanine at position 64 cause an increase in hydrophobicity in the N-terminus and active site loop thereby restricting water entry and disrupting the water network in the Zn2+-binding pocket. To pave the way for a detailed investigation into the structural, functional, and mechanistic aspects of the Ser2Ala/His64Ala double mutant of hCAII, we present here almost complete sequence-specific resonance assignments for 1H, 15N, and 13C. These assignments serve as the basis for comprehensive studies on the dynamics of the protein-water network within the Zn2+-binding pocket and its role in catalysis.

Plasmonic Nanotrenches with 1 nm Nanogaps for Surface-Enhanced Raman Scattering-Based Screening of His-Tagged Proteins.

This study represents a highly sensitive and selective approach to protein screening using surface-enhanced Raman scattering (SERS) facilitated by octahedral Au nanotrenches (OANTs). OANTs are a novel class of nanoparticles characterized by narrow, trench-like excavations indented into the eight facets of a Au octahedron. This unique configuration maximizes electromagnetic near-field focusing as the gap distance decreases to ∼1 nm. Owing to geometrical characteristics of the OANTs, near-field focusing can be maximized through the confinement and reflectance of light trapped within the trenches. We used Ni ions and molecular linkers to confer selective binding affinity for His-tagged proteins on the surfaces of the OANTs for SERS-based protein screening. Remarkably, SERS-based protein screening with the surface-modified OANTs yielded outstanding screening capabilities: 100% sensitivity and 100% selectivity in distinguishing His-tagged human serum albumin (HSA) from native HSA. This highlights the significantly enhanced protein screening capabilities achieved through the synergistic combination of SERS and the OANTs.