Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Expanding the Hydrophobic Cavity Surface of Azocalix[4]arene to Enable Biotin/Avidin Affinity with Controlled Release.

The development of artificial receptors that combine ultrahigh-affinity binding and controllable release for active guests holds significant importance in biomedical applications. On one hand, a complex with an exceedingly high binding affinity can resist unwanted dissociation induced by dilution effect and complex interferents within physiological environments. On the other hand, stimulus-responsive release of the guest is essential for precisely activating its function. In this context, we expanded hydrophobic cavity surface of a hypoxia-responsive azocalix[4]arene, affording Naph-SAC4A. This modification significantly enhanced its aqueous binding affinity to 1013 M-1, akin to the naturally occurring strongest recognition pair, biotin/(strept-)avidin. Consequently, Naph-SAC4A emerges as the first artificial receptor to simultaneously integrate ultrahigh recognition affinity and actively controllable release. The markedly enhanced affinity not only improved Naph-SAC4A's sensitivity in detecting rocuronium bromide in serum, but also refined the precision of hypoxia-responsive doxorubicin delivery at the cellular level, demonstrating its immense potential for diverse practical applications.

Macrophage membrane (MMs) camouflaged near-infrared (NIR) responsive bone defect area targeting nanocarrier delivery system (BTNDS) for rapid repair: promoting osteogenesis via phototherapy and modulating immunity.

Bone defects remain a significant challenge in clinical orthopedics, but no targeted medication can solve these problems. Inspired by inflammatory targeting properties of macrophages, inflammatory microenvironment of bone defects was exploited to develop a multifunctional nanocarrier capable of targeting bone defects and promoting bone regeneration. The avidin-modified black phosphorus nanosheets (BP-Avidin, BPAvi) were combined with biotin-modified Icaritin (ICT-Biotin, ICTBio) to synthesize Icaritin (ICT)-loaded black phosphorus nanosheets (BPICT). BPICT was then coated with macrophage membranes (MMs) to obtain MMs-camouflaged BPICT (M@BPICT). Herein, MMs allowed BPICT to target bone defects area, and BPICT accelerated the release of phosphate ions (PO43-) and ICT when exposed to NIR irradiation. PO43- recruited calcium ions (Ca2+) from the microenvironment to produce Ca3(PO4)2, and ICT increased the expression of osteogenesis-related proteins. Additionally, M@BPICT can decrease M1 polarization of macrophage and expression of pro-inflammatory factors to promote osteogenesis. According to the results, M@BPICT provided bone growth factor and bone repair material, modulated inflammatory microenvironment, and activated osteogenesis-related signaling pathways to promote bone regeneration. PTT could significantly enhance these effects. This strategy not only offers a solution to the challenging problem of drug-targeted delivery in bone defects but also expands the biomedical applications of MMs-camouflaged nanocarriers.

Live-cell imaging of human apurinic/apyrimidinic endonuclease 1 in the nucleus and nucleolus using a chaperone@DNA probe.

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.

Evaluación de reactividad inmunohistoquímica con método del Complejo Avidina­Biotina (ABC) / Evaluation of immunohistochemical reactivity with Avidin-Biotin Complex (ABC) method

Inmunohistoquímica es toda técnica que permite detectar in situ componentes celulares y extracelulares por medio de anticuerpos específicos, empleando sistemas de detección enzimáticos. Dentro de los métodos inmunohistoquímicos, la técnica del complejo avidina­biotina(ABC) es ampliamente utilizada debido a su alta sensibilidad. El objetivo del presente estudio fueevaluar la reactividad inmunohistoquímica del anticuerpo 4C4.9 para la detección de la proteínaS-100, utilizando el método ABC. Para la evaluación de la reactividad inmunohistoquímica se utilizaron 2 biopsias de piel humana con diagnóstico histopatológico de melanoma maligno nodular ulcerado y nevus melanocítico intradérmico, provenientes del Laboratorio de Investigación en Biotecnología Animal de la Universidad de La Frontera, Temuco, Chile. Se utilizó el Kit VECTASTAIN®como método de detección, la dilución del anticuerpo 4C4.9 fue 1/250 y la temperatura de incubación fue a 4 ºC ó 37 ºC por 18 horas. Para validar la técnica, se realizó un control positivo y otro negativo para 4C4.9. Los resultados de la tinción inmunohistoquímica por el método del complejo ABC mostraron tinción positiva para la proteína S-100, tanto en melanoma maligno nodular ulcerado, como en nevus melanocítico intradérmico, incubados durante 18 horas a 4 ºC ó 37 ºC. Sin embargo, la inmunotinción fue más intensa cuando el anticuerpo primario se incubó a 37 ºC. Para una correcta interpretación de los resultados, es necesario tener en consideración que la reacción antígeno-anticuerpo se ve influenciada por diversos factores, como la concentración del anticuerpo, el tiempo y la temperatura de incubación. En conclusión, nuestros resultados sugieren incubarlas muestras con el primer anticuerpo (4C4.9) en una dilución de 1/250 en agua destilada, incu-bando durante 18 h a 37 ºC. Se recomienda la utilización del anticuerpo 4C4.9 como apoyo al diagnóstico y diagnóstico diferencial.

Immunohistochemistry is anytechnique that can detect cellular and extracellular components in situ by means of specific antibodies,using enzymatic detection systems. Among immunohistochemical methods, the technique ofavidin - biotin complex (ABC) is widely used because of its high sensitivity. The aim of this study was to evaluate the immunohistochemical reactivity of the4C4.9 antibody for detection of S-100 protein using the ABC method. For the evaluation ofimmunohistochemical reactivity 2 biopsies of humanskin were used with histopathological diagnosis ofulcerated malignant melanoma and melanocyticintradermal nevi from the Research Laboratory onAnimal Biotechnology of the Universidad de La Fron-tera, Chile. The Kit VECTASTAIN® was used asdetection method, the dilution the 4C4.9 antibodywas 1/250 and incubation temperature was at 4 °Cor 37 °C for 18 hours. To validate the technique, apositive control and a negative for 4C4.9 was performed. The results of immunohistochemicalstaining by the method of ABC complex showed positive staining for protein S-100 both in ulcerated malignant melanoma and melanocytic intradermalnevi, incubated for 18 hours at 4 °C or 37 °C.However, immunostaining was more intense when the primary antibody was incubated at 37° C. For acorrect interpretation of the results, it is necessary to take into consideration that the antigen-antibody reaction is influenced by various factors such as the concentration of antibody, time and temperature ofincubation. In conclusion, our results suggest incubating the samples with the first antibody (4C4.9)at 1/250 dilution in distilled water, incubating for 18h at 37 ºC. However, immunostaining was moreintense when the primary antibody was incubated at37° C. For a correct interpretation of the results, it isnecessary to take into consideration that antigen-antibody reaction is influenced by various factors suchas the concentration of antibody, time and temperature of incubation. In conclusion, our results suggest incubating the samples with the first antibody(4C4.9) at 1/250 dilution in distilled water, incubating for 18 h at 37 ºC. The use of the antibody 4C4.9 is recommended to support the diagnosis and differential diagnosis.