Novel Polystyrene-Binding Nanobody for Enhancing Immunoassays: Insights into Affinity, Immobilization, and Application Potential.

Nanobodies, which represent the next generation of antibodies due to their unique properties, face a significant limitation in their poor physical adsorption on solid supports. In this study, we successfully discovered polystyrene binding nanobodies from a synthetic nanobody library. Notably, bivalent nanobody B2 exhibited high affinity for polystyrene (0.7 nM for ELISA saturation binding analysis and 15.6 nM for isothermal titration calorimetry), displaying a pH-dependent behavior. Remarkably, hydrophobic and electrostatic interactions contribute minimally to the binding process. Molecular modeling provided insights into the interaction between B2 and polystyrene, revealing that the Trp51 residue within the CDR2 loop formed an aromatic H-bond with polystyrene at a distance of 2.74 Å, thus explaining the observed reduction in B2 affinity caused by Trp51 mutations. To explore B2's potential in protein immobilization, we constructed a bispecific nanobody by fusing B2 to an anticarcinoembryonic antigen nanobody 11C12, which cannot be immobilized on polystyrene through passive adsorption. Remarkably, the fusion construct achieved effective immobilization on polystyrene within 5 min by passing the need for periplasmic protein purification despite its low expression level. Moreover, the fusion construct demonstrated excellent linearity in the chemiluminescent enzyme immunoassay. For the first time, this study reports a simplified and seamless platform for the oriented immobilization of nanobody. Importantly, the entire process eliminated the need for protein purification, enabling efficient and rapid immobilization of fusion proteins directly from crude cell extracts, even when the expression level was low. Our developed process dramatically reduced the processing time from 2.5 days to just 5 min.

Optimized silk fibroin nanoparticle functionalization with anti-CEA nanobody enhancing active targeting of colorectal cancer cells.

This work aimed to establish a simple and feasible method to obtain silk fibroin nanoparticles (SFNPs) with uniform particles size, and then modify the SFNPs with nanobody (Nb) 11C12 targeting the proximal membrane end of carcinoembryonic antigen on the surface of colorectal cancer (CRC) cells. The regenerated silk fibroin (SF) was isolated using ultrafiltration tubes with a 50 kDa molecular weight cut-off, and the retention fraction (named as SF > 50 kDa) was further self-assembled into SFNPs by ethanol induction. Scanning electron microscope (SEM) and high-resolution transmission electron microscop showed that the SFNPs with uniform particles size were formed. Due to electrostatic adsorption and pH responsiveness, SFNPs have been proved to effectively load and release the anticancer drug doxorubicin hydrochloride (DOX) (DOX@SFNPs). Further, targeting molecule Nb 11C12 was used to modify these nanoparticles, constituting the targeted outer layer of the drug delivery system (DOX@SFNPs-11C12), achieving precise localization to cancer cells. The release amount of DOX observed fromin vitrodrug release profiles increased as follows: pH 7.4 < pH 6.8 < pH 5.4, demonstrating that the DOX release could be accelerated in a weakly acidic environment.In vitrocytotoxicity experiments displayed that SFNPs-11C12 nanoparticles exhibited good safety and biocompatibility. Drug-loaded nanoparticles, DOX@SFNPs-11C12, led to higher LoVo cells apoptosis compared to DOX@SFNPs. Fluorescence spectrophotometer characterization and confocal laser scanning microscopy further showed that the internalization of DOX was highest in the DOX@SFNPs-11C12, certifying that the introduced targeting molecule enhanced the uptake of drug delivery system by LoVo cells. This study provides a simple and operational approach to developing an optimized SFNPs drug delivery system modified by targeting Nb, which can be a good candidate for CRC therapy.

Minimal non-abelian nodal braiding in ideal metamaterials.

Exploring new topological phases and phenomena has become a vital topic in condensed matter physics and materials sciences. Recent studies reveal that a braided colliding nodal pair can be stabilized in a multi-gap system with [Formula: see text] or [Formula: see text] symmetry. This exemplifies non-abelian topological charges beyond the scope of conventional single-gap abelian band topology. Here, we construct ideal acoustic metamaterials to realize non-abelian braiding with the fewest band nodes. By emulating the time with a sequence of acoustic samples, we experimentally observe an elegant but nontrivial nodal braiding process, including nodes creation, braiding, collision, and repulsion (i.e., impossible to annihilate), and measure the mirror eigenvalues to elucidate the braiding consequence. The latter, at the level of wavefunctions, is of prime importance since essentially braiding physics aims to entangle multi-band wavefunctions. Furthermore, we experimentally unveil the highly intricate correlation between the multi-gap edge responses and the bulk non-abelian charges. Our findings pave the way for developing non-abelian topological physics that is still in its infancy.

Observation of Acoustic Non-Hermitian Bloch Braids and Associated Topological Phase Transitions.

Topological features embedded in ancient braiding and knotting arts endow significant impacts on our daily life and even cutting-edge science. Recently, fast growing efforts are invested to the braiding topology of complex Bloch bands in non-Hermitian systems. This new classification of band topology goes far beyond those established in Hermitian counterparts. Here, we present the first acoustic realization of the topological non-Hermitian Bloch braids, based on a two-band model easily accessible for realizing any desired knot structure. The non-Hermitian bands are synthesized by a simple binary cavity-tube system, where the long-range, complex-valued, and momentum-resolved couplings are accomplished by a well-controlled unidirectional coupler. In addition to directly visualizing various two-band braiding patterns, we unambiguously observe the highly elusive topological phase transitions between them. Not only do our results provide a direct demonstration for the non-Hermitian band topology, but also the experimental techniques open new avenues for designing unconventional acoustic metamaterials.

Tracking Valley Topology with Synthetic Weyl Paths.

Inspired by the newly emergent valleytronics, great interest has been attracted to the topological valley transport in classical metacrystals. The presence of nontrivial domain-wall states is interpreted with a concept of valley Chern number, which is well defined only in the limit of small band gap. Here, we propose a new visual angle to track the intricate valley topology in classical systems. Benefiting from the controllability of our acoustic metacrystals, we construct Weyl points in synthetic three-dimensional momentum space through introducing an extra structural parameter (rotation angle here). As such, the two-dimensional valley-projected band topology can be tracked with the strictly quantized topological charge in three-dimensional Weyl crystal, which features open surface arcs connecting the synthetic Weyl points and gapless chiral surface states along specific Weyl paths. All theoretical predictions are conclusively identified by our acoustic experiments. Our findings may promote the development of topological valley physics, which is less well defined yet under hot debate in multiple physical disciplines.

Acoustic Realization of Surface-Obstructed Topological Insulators.

Recently, higher-order topological insulators have been attracting extensive interest. Unlike the conventional topological insulators that demand bulk gap closings at transition points, the higher-order band topology can be changed without bulk closure and exhibits as an obstruction of higher-dimensional boundary states. Here, we report the first experimental realization of three-dimensional surface-obstructed topological insulators with using acoustic crystals. Our acoustic measurements demonstrate unambiguously the emergence of one-dimensional topological hinge states in the middle of the bulk and surface band gaps, as a direct manifestation of the higher-order band topology. Together with comparative measurements for the trivial and phase-transition-point insulators, our experimental data conclusively evidence the unique bulk-boundary physics for the surface-obstructed band topology. That is, the topological phase transition is determined by the closure of the surface gap, rather than by closing the bulk gap. Our study might spur on new activities to deepen the understanding of such elusive topological phases.

Acoustic Möbius Insulators from Projective Symmetry.

In the presence of gauge symmetry, common but not limited to artificial crystals, the algebraic structure of crystalline symmetries needs to be projectively represented, giving rise to unprecedented topological physics. Here, we demonstrate this novel idea by exploiting a projective translation symmetry and constructing a variety of Möbius-twisted topological phases. Experimentally, we realize two Möbius insulators in acoustic crystals for the first time: a two-dimensional one of first-order band topology and a three-dimensional one of higher-order band topology. We observe unambiguously the peculiar Möbius edge and hinge states via real-space visualization of their localiztions, momentum-space spectroscopy of their 4π periodicity, and phase-space winding of their projective translation eigenvalues. Not only does our work open a new avenue for artificial systems under the interplay between gauge and crystalline symmetries, but it also initializes a new framework for topological physics from projective symmetry.

A novel silk fibroin protein-based fusion system for enhancing the expression of nanobodies in Escherichia coli.

Nanobodies show a great potential in biomedical and biotechnology applications. Bacterial expression is the most widely used expression system for nanobody production. However, the yield of nanobodies is relatively low compared to that of eukaryotic systems. In this study, the repetitive amino acid sequence motifs (GAGAGS) found in silk fibroin protein (SFP) were developed as a novel fusion tag (SF-tag) to enhance the expression of nanobodies in Escherichia coli. SF-tags of 1 to 5 hexapeptide units were fused to the C-terminus of 4G8, a nanobody against human epididymis protein 4 (HE4). The protein yield of 4G8 variants was increased by the extension of hexapeptide units and achieved a 2.5 ~ 7.1-fold increase compared with that of untagged 4G8 (protein yield of 4G8-5C = 0.307 mg/g vs that of untagged 4G8 = 0.043 mg/g). Moreover, the fusion of SF-tags not only had no significant effect on the affinity of 4G8, but also showed a slight increase in the thermal stability of SF-tag-fused 4G8 variants. The fusion of SF-tags increased the transcription of 4G8 by 2.3 ~ 7.0-fold, indicating SF-tags enhanced the protein expression at the transcriptional level. To verify the applicability of the SF-tags for other nanobody expression, we further investigated the protein expression of two other anti-HE4 nanobodies 1G8 and 3A3 upon fusion with the SF-tags. Results indicated that the SF-tags enhanced the protein expression up to 5.2-fold and 5.7-fold for 1G8 and 3A3, respectively. For the first time, this study reported a novel and versatile fusion tag system based on the SFP for improving nanobody expression in Escherichia coli, which may enhance its potential for wider applications.Key points• A silk fibroin protein-based fusion tag (SF-tag) was developed to enhance the expression of nanobodies in Escherichia coli.• The SF-tag enhanced the nanobody expression at the transcriptional level.• The fusion of SF-tag had no significant effect on the affinity of nanobodies and could slightly increase the thermal stability of nanobodies.

Targeting and neutralizing human epididymis protein 4 by novel nanobodies to suppress ovarian cancer cells and attenuate cisplatin resistance.

Human epididymis protein 4 (HE4) is a glycoprotein secreted by epithelial ovarian cancer (EOC) cells and is a novel and specific biomarker for diagnosing and prognosing EOC. Previous studies have shown that overexpression of HE4 is correlated with EOC tumorigenesis and chemoresistance. However, less has been reported regarding the direct effect of the secreted HE4 protein as an autocrine factor in EOC cells. Here, we investigated the molecular mechanism of the secretory form of HE4 on the growth of EOC cells by applying nanobodies with a targeted interaction of free HE4. Three anti-HE4 nanobodies were selected from an immune library by phage display. HE4 secreted by serum-free cultured OVCAR3 cells increased and was effectively neutralized by anti-HE4 nanobodies, which inhibited cell viability. Treatment with the anti-HE4 nanobody 1G8 decreased Bcl-2 expression and increased BAX, cleaved PARP, and p53 levels, resulting in apoptosis of OVCAR3 cells. Moreover, 1G8 significantly improved the cisplatin response of OVCAR3 cells. Our data suggest that secretory HE4 played a novel pro-survival autocrine role and was a target of the anti-HE4 nanobody to improve the therapeutic effects of cisplatin-based chemotherapy.

In Situ Maturation and Tissue Adaptation of Type 2 Innate Lymphoid Cell Progenitors.

Innate lymphoid cells (ILCs) are generated early during ontogeny and persist predominantly as tissue-resident cells. Here, we examined how ILCs are maintained and renewed within tissues. We generated a single cell atlas of lung ILC2s and found that Il18r1+ ILCs comprise circulating and tissue-resident ILC progenitors (ILCP) and effector-cells with heterogeneous expression of the transcription factors Tcf7 and Zbtb16, and CD103. Our analyses revealed a continuous differentiation trajectory from Il18r1+ ST2- ILCPs to Il18r- ST2+ ILC2s, which was experimentally validated. Upon helminth infection, recruited and BM-derived cells generated the entire spectrum of ILC2s in parabiotic and shield chimeric mice, consistent with their potential role in the renewal of tissue ILC2s. Our findings identify local ILCPs and reveal ILCP in situ differentiation and tissue adaptation as a mechanism of ILC maintenance and phenotypic diversification. Local niches, rather than progenitor origin, or the developmental window during ontogeny, may dominantly imprint ILC phenotypes in adult tissues.

Development of tilapia collagen and chitosan composite hydrogels for nanobody delivery.

Recently, injectable hydrogels have shown great potential in cell therapy and drug delivery. They can easily fill in any irregular-shaped defects and remain in desired positions after implantation using minimally invasive strategies. Here, we developed hydrogels prepared from tilapia skin collagen and chitosan (HCC). The residual mass rate of HCC was affected by the pH at the time of preparation, which was 29.1 % at pH 7 in 36 h. By comparison, the residual mass ratios of HCC at pH values of 6 and 5 were only approximately 8.4 % and 0, respectively. In addition, the stability of HCC was also affected by the concentration of these two components. HCC10 catalyzed by 10 mg mL-1 tilapia skin collagen and 10 mg mL-1 chitosan was more stable than HCC5 catalyzed by 5 mg mL-1 tilapia skin collagen and 10 mg mL-1 chitosan; therefore, we studied that ability of HCC10 to deliver two model nanobodies: 2D5 and KPU. As the concentration of nanobodies increased, the cumulative release rate of 2D5 decreased, and the release rate of KPU increased. Meanwhile, the cumulative release rate of 2D5 was the highest (68.3 %) at pH 5.5, followed by pH 6.8 (56.4 %) and 7.4 (28.4 %). However, the cumulative release rates of KPU were similar at pH 5.5 (45.1 %), 6.8 (46.5 %), and 7.4 (44.9 %). HCC is biodegradable, and can facilitate the release nanobodies; thus, HCC could be developed into an intelligent responsive tumor treatment matrix for use in cancer therapy.

Development and characterization of a photo-cross-linked functionalized type-I collagen (Oreochromis niloticus) and polyethylene glycol diacrylate hydrogel.

Collagen hydrogels have been widely investigated as scaffolds for tissue engineering due to their biocompatibility and capacity to promote cell adhesion. However, insufficient mechanical strength and rapid degradation properties remain the major obstacles for their applications. In the present study, type-I tilapia collagen (TC) was functionalized to form methacrylated tilapia collagen (MATC) by introducing methacrylic acid, developing a photo-cross-linked PEGDA-MATC hydrogel. The mechanical strength of PEGDA-MATC hydrogel could be tuned by adjusting the pH of the precursor solutions, which was decreased with the pH increased. At a pH 5 condition, PEGDA-MATC showed the highest compressive fracture stress (1.31 MPa). Compared to the PEGDA-TC hydrogel, PEGDA-MATC hydrogel exhibited similar swelling behavior to PEGDA-TC hydrogel in PBS solutions, but higher residual mass ratio (PEGDA-MATC, 213.2 ± 2.8%) than PEGDA-TC hydrogel (199.4 ± 3.8%) when cultured with type-I collagenase. PEGDA-MATC hydrogel showed sustained BSA release capacity for 6 days, and the BSA release ratio was significantly (p < 0.05) decreased with increasing concentration of loaded-BSA (68.6% at 4 mg mL-1, 42.2% at 8 mg mL-1). The PEGDA-MATC hydrogel allowed cell adhesion and proliferation in vitro. These results demonstrated that PEGDA-MATC hydrogel might be a potential scaffold for tissue engineering applications.

Tracing the Equilibrium Phase of Cancer Immunoediting in Epidermal Neoplasms via Longitudinal Intravital Imaging.

Recognition of transformed cells by the immune system can sometimes generate a rate-limiting equilibrium phase, wherein tumor outgrowth is prevented without complete neoplasm elimination. Targeting premalignancies during this immune-controlled bottleneck is a promising strategy for rational cancer prevention. Thus far, immune equilibrium has been difficult to model in a traceable way, and most immunoediting systems have been limited to mesenchymal tumor types. Here, we introduce a mouse model for fluorescent tracing of somatic epithelial transformation. We demonstrate that transplantation can be used to prevent a confounding artificial tolerance that affects autochthonous inducible models. Using this system, we observe the expected dichotomy of outcomes: immune-deficient contexts permit rapid tumorigenesis, whereas initiated clones in immunocompetent mice undergo elimination or equilibrium. The equilibrium phase correlates with localization within hair follicles, which have been characterized previously as relatively immune-privileged sites. Given this, we posit that valleys in the immune surveillance landscape of a normal tissue can provide a cell-extrinsic alternative to the canonical cell-intrinsic adaptations believed to establish the equilibrium phase. Our model is a prototype for tracing immunoediting in vivo and could serve as a novel screening platform for therapies targeted against immune-controlled premalignancies.

A basal-enriched microRNA is required for prostate tumorigenesis in a Pten knockout mouse model.

MicroRNAs (miRNAs) play important roles in prostate cancer development. However, it remains unclear how individual miRNAs contribute to the initiation and progression of prostate cancer. Here we show that a basal layer-enriched miRNA is required for prostate tumorigenesis. We identify miR-205 as the most highly expressed miRNA and enriched in the basal cells of the prostate. Although miR-205 is not required for normal prostate development and homeostasis, genetic deletion of miR-205 in a Pten null tumor model significantly compromises tumor progression and does not promote metastasis. In Pten null basal cells, loss of miR-205 attenuates pAkt levels and promotes cellular senescence. Furthermore, although overexpression of miR-205 in prostate cancer cells with luminal phenotypes inhibits cell growth in both human and mouse, miR-205 has a minimal effect on the growth of a normal human prostate cell line. Taken together, we have provided genetic evidence for a requirement of miR-205 in the progression of Pten null-induced prostate cancer.

Probing Weyl Physics with One-Dimensional Sonic Crystals.

Recently, intense efforts have been devoted to realizing classical analogues of various topological phases of matter. In this Letter, we explore the intriguing Weyl physics by a simple one-dimensional sonic crystal, in which two extra structural parameters are combined to construct a synthetic three-dimensional space. Based on our ultrasonic experiments, we have not only observed the synthetic Weyl points, but also probed the novel reflection phase singularity that connects inherently with the topological robustness of Weyl points. The presence of topologically nontrivial interface modes has been demonstrated further. As the first realization of topological acoustics in synthetic space, our study exhibits great potential of probing high-dimensional topological phenomena by such easily fabricated and detected low-dimension acoustic systems.

CD49b defines functionally mature Treg cells that survey skin and vascular tissues.

Regulatory T (Treg) cells prevent autoimmunity by limiting immune responses and inflammation in the secondary lymphoid organs and nonlymphoid tissues. While unique subsets of Treg cells have been described in some nonlymphoid tissues, their relationship to Treg cells in secondary lymphoid organs and circulation remains unclear. Furthermore, it is possible that Treg cells from similar tissue types share largely similar properties. We have identified a short-lived effector Treg cell subset that expresses the α2 integrin, CD49b, and exhibits a unique tissue distribution, being abundant in peripheral blood, vasculature, skin, and skin-draining lymph nodes, but uncommon in the intestines and in viscera-draining lymph nodes. CD49b+ Treg cells, which display superior functionality revealed by in vitro and in vivo assays, appear to develop after multiple rounds of cell division and TCR-dependent activation. Accordingly, single-cell RNA-seq analysis placed these cells at the apex of the Treg developmental trajectory. These results shed light on the identity and development of a functionally potent subset of mature effector Treg cells that recirculate through and survey peripheral tissues.

Single Cell and Open Chromatin Analysis Reveals Molecular Origin of Epidermal Cells of the Skin.

How embryonic progenitors coordinate cell fate specification and establish transcriptional and signaling competence is a fundamental question in developmental biology. Here, we show that transcription factor ΔNp63 profoundly changes the transcriptome and remodels thousands of open chromatin regions of Krt8+ progenitors during epidermal fate specification. ATAC-seq and single-cell RNA-seq reveal that ΔNp63-dependent programs govern epidermal lineage formation, and ΔNp63-independent programs, mediated by AP2 and AP1 transcription factors, promote epidermal differentiation and epithelial-to-mesenchymal transition. ΔNp63 promotes Wnt signaling by directly upregulating Wnt ligands, Frizzled receptors, and transcription factors. Deletion of ß-catenin in Krt8+ progenitors delays their maturation into Krt5+ progenitors. The lack of epidermal Wnt production in the absence of ΔNp63 also incapacitates Wnt activation in the underlying dermal cells. These findings reveal the remarkable changes of the transcriptome, open chromatin, and signaling pathways at the onset of skin development and uncover the molecular cascade for epidermal lineage formation.

Topological negative refraction of surface acoustic waves in a Weyl phononic crystal.

Reflection and refraction of waves occur at the interface between two different media. These two fundamental interfacial wave phenomena form the basis of fabricating various wave components, such as optical lenses. Classical refraction-now referred to as positive refraction-causes the transmitted wave to appear on the opposite side of the interface normal compared to the incident wave. By contrast, negative refraction results in the transmitted wave emerging on the same side of the interface normal. It has been observed in artificial materials1-5, following its theoretical prediction6, and has stimulated many applications including super-resolution imaging7. In general, reflection is inevitable during the refraction process, but this is often undesirable in designing wave functional devices. Here we report negative refraction of topological surface waves hosted by a Weyl phononic crystal-an acoustic analogue of the recently discovered Weyl semimetals8-12. The interfaces at which this topological negative refraction occurs are one-dimensional edges separating different facets of the crystal. By tailoring the surface terminations of the Weyl phononic crystal, constant-frequency contours of surface acoustic waves can be designed to produce negative refraction at certain interfaces, while positive refraction is realized at different interfaces within the same sample. In contrast to the more familiar behaviour of waves at interfaces, unwanted reflection can be prevented in our crystal, owing to the open nature of the constant-frequency contours, which is a hallmark of the topologically protected  surface states in Weyl crystals8-12.