Search

Background: Excessive aggregation of β-amyloid peptides (Aβ) is regarded as the hallmark of Alzheimer’s disease. Exploring the underlying mechanism regulating Aβ aggregation remains challenging and investigating aggregation events of Aβ in the presence and absence of metal ions at molecular level would be meaningful in elucidating the role of metal cations on interactions between Aβ molecules. In this study, chemical self-assembled monolayer (SAM) method was employed to fabricate monolayer of β-amyloid peptides Aβ42 on gold substrate with a bolaamphiphile named 16-Mercaptohexadecanoic acid (MHA). Firstly, the samples of gold substrate (blank control), the MHA-modified substrate and the Aβ42-modified substrate were detected by X-ray photoelectron spectroscopy (XPS) to track the self-assembly process. Aggregation behaviors of Aβ42 before and after metallic ions (Zn2+、Ca2+、Al3+) treated were monitored by atomic force microscopy (AFM) and the interaction between Aβ42 and metallic ions (Zn2+、Ca2+、Al3+) was investigated by surface-enhanced Raman Scattering (SERS), respectively.Results: The XPS spectra of binding energy of gold substrate (blank control), the MHA-modified substrate and the Aβ42-modified substrate are well fitted with the corresponding monolayer’s composition, which indicates that Aβ42 monolayer is well formed. The recorded surface morphology of different experimental groups obtained by AFM showed markedly different nanostructures, indicating occurrence of aggregation events between Aβ42 molecules after adding metal ions to the solution. Compared to the control group, the presence of metal ions resulted in the increased size of surface structures on the observed 3D topography. Further study by SERS showed that the Raman strength of Aβ42 changes significantly after the metal cation treatment. A considerable part of the amide bonds interacts with metal cations, leading to a structural change, which is characterized by the weakened β-fold Raman peak.Conclusion: The AFM imaging results suggest that aggregation events occurred between Aβ42 molecules with the addition of metal cations. Furthermore, the effect of metallic cations on the conformational change of Aβ42 studied by SERS supported the results obtained by AFM imaging. Taken together, the results showed that the presence of substoichiometric metal cations promotes aggregation behavior between Aβ42 molecules on the substrate at pH 7.4.

Abstract: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death, which can be attributed to the high incidence and first diagnosis at an advanced stage. Tyrosine kinase inhibitors (TKIs), a class of small-molecule targeting drugs, are primarily used for the clinical treatment of HCC after chemotherapy because they show significant clinical efficacy and low incidence of clinical adverse reactions. However, resistance to sorafenib and other TKIs, which can be used to treat advanced HCC, poses a significant challenge. Recent mechanistic studies have shown that epithelial-mesenchymal transition or transformation (EMT), ATP binding cassette (ABC) transporters, hypoxia, autophagy, and angiogenesis are involved in apoptosis, angiogenesis, HCC cell proliferation, and TKI resistance in patients with HCC. Exploring and overcoming such resistance mechanisms is essential to extend the therapeutic benefits of TKIs to patients with TKI-resistant HCC. This review aims to summarize the potential resistance mechanism proposed in recent years and methods to reverse TKI resistance in the context of HCC.

In this study, self-assembled monolayer (SAM) method was employed to fabricate monolayer of β-amyloid peptides Aβ42 on gold substrate with a bolaamphiphile named thiol (MHA). Firstly, the samples of gold substrate (blank control), the MHA-modified substrate and the Aβ42-modified substrate were detected by X-ray photoelectron spectroscopy (XPS) to track the self-assembly process. The spectra of binding energy measured from these three sample surfaces could be well fitted with the corresponding monolayer’s composition, which means Aβ42 monolayer is well formed. Aggregation behaviors of Aβ42 in the absence and presence of metallic ions (Zn2+、Ca2+、Al3+) were then monitored by atomic force microscopy (AFM) and surface-enhanced Raman Scattering (SERS), respectively. The recorded surface morphology of different experimental groups obtained by AFM showed markedly different nanostructures, indicating occurrence of aggregation behaviors of Aβ42. In solutions with added metal ions, the increased size of surface structures was observed, which suggest the presence of metal cations promotes aggregation behavior between Aβ42 molecules. Furtherly, the interaction between Aβ42 and metal cations was investigated by SERS. The results demonstrate that the Raman strength of Aβ42 changes after the metal cation treatment. Taken together, the combined AFM imaging and Raman analyses show that the three kinds of metallic ions promote the process of Aβ42 aggregation.

Characterization and determination of protein-protein interactions (PPIs) plays an important role in molecular biological science. In this study, the effect of aminoglycosides (AGs: streptomycin, gentamycin, lincomycin and clindamycin) on interactions between bovine serum albumin (BSA) was evaluated employing imaging and probing adhesion event by AFM. Multi-spectroscopy and molecular docking were supplementary to investigate the acting forces of the effect. AFM measurements revealed the aggregation of BSA grains and changes of adhesion forces at single molecule level. With adhesion forces between BSA pairs decomposed by Poisson method, specific forces in streptomycin, gentamycin, lincomycin and climdamycin were obviously decreased with the rate of 33.1%, 26.4%, 32.3% and 31.3% while non-specific forces slightly decreased with 5.5%, 3.3%, 4.0% and 7.7%. Combined with results of multi-spectroscopy as well as molecular docking, the whole determination showed AGs affected PPIs by multiple forces, where the hydrogen bonding and hydration effect were the main reasons. The binding of drugs and proteins acted by hydrogen bonding affected the interaction forces between BSA. Consequently, AFM was proposed to be an effective and precise tool in application including evaluating the effects of exogenous compounds on biomacromolecular interactions and rapid screening of drug candidates to avoid potential damages in disease treatment.

Acyclovir (ACV), an analog of 2′-deoxyguanosine, exhibits poor bioavailability due to limited permeability. In this study, the aim was to improve the bioavailability and permeability of ACV. N,N,N-trimethyl-O-octadecyl chitosan (TMSC) was synthesized by covalently bonding a long alkyl chain and a quaternary ammonium group onto chitosan. The chemical structure of TMSC was characterized by Fourier transform infrared (FT-IR) spectra, proton nuclear magnetic resonance (1H-NMR), thermogravimetry (TG), and differential thermogravimetry (DTG). The biological safety of TMSC was evaluated using a hemolysis experiment, which resulted in a hemolysis rate of 6.63% at the highest concentration (3 mg/mL). TMSC was used to prepare acyclovir nanoparticle (ACV NP) based on the Box–Behnken design, and characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS) to determine size and morphology. Furthermore, the permeability of ACV NP was investigated using a non-everted intestinal sac model. ACV NP exerted a 1.9-fold higher effect on the ileum than ACV. In vivo, the relative bioavailability of ACV NP was ∼4.5-fold greater than that of the ACV suspension. These results suggest that ACV NP augment permeability and effectiveness of ACV.

Since the advances in protein engineering and manufacture, over the last 30 years, antibody-based immunotherapeutic has become a powerful strategy to treat diseases. The T-cell engaging bispecific antibody (BsAb) by combining the Fab binding domain of tumor antigens and Fab or single-chain variable fragments (scFvs) binding domain of CD3 molecules, could redirect cytotoxic T cells to kill tumor cells. The IgG-scFv format of BsAb is a dual bivalent and asymmetrical design, which adds the benefit of potent cytotoxicity and less complicated for manufacture but limits the stability and production. Here, we engineered a series of interchain disulfide bonds in the Fab region of IgG-svFv BsAbs and evaluated its biophysical and biological properties. We found that simultaneously replaced the position of VH44-VL100 and CH1126-CL121 residues with cysteine, to form two additional disulfide bonds, could markedly increase monomeric BsAb formation and yield. The thermostability and stability against aggregation and degradation also performed better than BsAbs without extra disulfide bonds introduction. Besides, the affinity of engineered BsAbs was maintained, and the h8B-BsAb antibody had a slight enhancement in an inhibitory effect on target cells.

BackgroundCancer stem cells (CSCs) and Circulating tumor cells (CTCs) have been proposed as fundamental causes for the recurrence of hepatocellular carcinoma (HCC). CTCs isolated from patients with HCC illustrate a unique Nanog expression profile analysis. The aim of this study was to enhance the prediction of recurrence and prognosis of the CTC phenotype in patients with HCC by combining Nanog expression into a combined forecasting model.Subjects, Materials, and MethodsWe collected 320 blood samples from 160 patients with HCC cancer before surgery and used CanPatrol™ CTC enrichment technology and in situ hybridization (ISH) to enrich and detect CTCs and CSCs. Nanog expression in all CTCs was also determined. In addition, RT-PCR and immunohistochemistry were used to study the expression of Nanog, E-Cadherin, and N-Cadherin in liver cancer tissues and to conduct clinical correlation studies.ResultsThe numbers of EpCAM mRNA+ CTCs and Nanog mRNA+ CTCs were strongly correlated with postoperative HCC recurrence (CTC number (P = 0.03), the total number of mixed CTCS (P = 0.02), and Nanog> 6.7 (P = 0.001), with Nanog > 6.7 (P = 0.0003, HR = 2.33) being the most crucial marker. There are significant differences in the expression of Nanog on different types of CTC: most Epithelial CTCs do not express Nanog, while most of Mixed CTC and Mesenchymal CTC express Nanog, and their positive rates are 38.7%, 66.7%, and 88.7%, respectively, (P=0.0001). Moreover, both CTC (≤/> 13.3) and Nanog (≤/>6.7) expression were significantly correlated with BCLC stage, vascular invasion, tumor size, and Hbv-DNA (all P ConclusionsThe number of Nanog-positive cells showed positive correlation with the poor prognosis of HCC patients. The detection and analysis of CTC markers (EpCAM and CK8, 18, CD45 Vimentin,Twist and 19) and CSCs markers (NANOG) are of great value in the evaluation of tumor progression.

Cancer therapies benefit from accelerated development of biotechnology, and many immunotherapeutic strategies spring up including vaccines, the immune checkpoint blockade, chimeric antigen receptor T cells, and bispecific antibodies (BsAbs). Glypican-3 (GPC3) is a member of the heparan sulfate proteoglycan family of proteins and is highly expressed in hepatocellular carcinoma (HCC) cell membranes. Here, the authors describe a new tetravalent BsAb h8B-BsAb targeting GPC3 and CD3 antigens and studied its antitumor activities against HCC. h8B-BsAb was designed based on immunoglobulin G with a fragment variable fused to the light chain, whose biophysical stabilities including degradation resistance and thermostability were improved by introducing disulfide bonds. In vitro activity of h8B-BsAb showed potent T-cell recruitment and activation for HCC cell lysis by the presence of peripheral blood mononuclear cells, but no specific killing in GPC3-negative cells. In HCC xenograft mouse studies, h8B-BsAb induced robust regression of tumors. In summary, we engineered a highly stable and efficacious BsAb as a potential candidate for HCC treatment.

Mechanobiology studies the means by which physical forces and mechanical properties change intra- or inter- biological macromolecules. Calmodulin (CaM) is involved in physiological activities and various metabolic processes in eukaryotic cells. Although the configuration changes in the interaction between calmodulin and melittin have been studied, the biomechanical relationship of their interaction has rarely been explored. Here, we measured the adhesion forces between calmodulin and melittin in solutions of gradient concentration of calcium ions using atomic force microscopy (AFM). We found that the specific (Fi) and nonspecific (F0) adhesion forces between single melittin and calmodulin in a PBS solution were 69.4 &plusmn, 5.0 and 29.3 &plusmn, 8.9 pN, respectively. In the presence of 10&minus, 7 to 10&minus, 3 M Ca2+ PBS solution, the Fi increased significantly to 93.8 &plusmn, 5.0, 139.9 &plusmn, 9.0, 140.4 &plusmn, 9.7, 171.5 &plusmn, 9.0, and 213.3 &plusmn, 17.8 pN, indicating that the unbinding force between melittin and calmodulin increased in the presence of Ca2+ in a concentration-dependent manner. These findings demonstrated that biomechanical studies based on AFM could help us better understand the melittin/calmodulin-binding processes in the presence of calcium and help us design and screen peptide drugs based on calmodulin.