Three Stages of Dynamic Assembly Process of Dipeptide-Based Supramolecular Gel Revealed by In Situ Infrared Spectroscopy

The exploration of short peptide-based assembly is vital for understanding protein-misfolding-associated diseases and seeking strategies to attenuate aggregate formation. While, the molecular mechanism of their structural evolution remains poorly studied in view of the dynamic and unpredictable assembly process. Herein, infrared (IR) spectroscopy, which serves as an in situ and real-time analytical technique, was intelligently employed to investigate the mechanism of phase transition and aggregate formation during the dynamic assembly process of diphenylalanine. Combined with other spectroscopy and electron microscopy technologies, three stages of gel formation and the main driving forces in different stages were revealed. A variety of stoichiometric methods such as continuous wavelet transform, principal component analysis, and two-dimensional correlation spectroscopy techniques were conducted to analyze the original time-dependent IR spectra to obtain detailed information on the changes in the amide bands and hydration layer. The microenvironment of hydrogen bonding among amide bands was significantly changed with the addition of pyridine derivatives, resulting in great differences in the properties of co-assembled gels. This work not only provides a universal analytical way to reveal the dynamic assembly process of dipeptide-based supramolecular gel but also expands their applications in supramolecular regulation and high-throughput screens in situ

Indirect Interlayer Bonding in Graphene–Topological Insulator van der Waals Heterostructure: Giant Spin–Orbit Splitting of the Graphene Dirac States

van der Waals (vdW) heterostructures of two-dimensional materials exhibit properties and functionalities that can be tuned by stacking order and interlayer coupling. Although direct covalent bonding is not expected at the heterojunction, the formation of an interface nevertheless breaks the symmetries of the layers, and the orthogonal requirement of the wave functions can lead to indirect interfacial coupling, creating new properties and functionalities beyond their constituent layers. Here, we fabricate graphene/topological insulator vdW heterostructure by transferring chemical vapor deposited graphene onto Bi₂Se₃ grown by molecular beam epitaxy. Using scanning tunneling microscopy/spectroscopy, we observe a giant spin–orbit splitting of the graphene Dirac states up to 80 meV. Density functional theory calculations further reveal that this splitting of the graphene bands is a consequence of the breaking of inversion symmetry and the orthogonalization requirement on the overlapping wave functions at the interface, rather than simple direct bonding. Our findings reveal two intrinsic characteristicsthe symmetry breaking and orthogonalization of the wave functions at the interfacethat underlines the properties of vdW heterostructures

Amplification of CD20 Cross-Linking in Rituximab-Resistant B‑Lymphoma Cells Enhances Apoptosis Induction by Drug-Free Macromolecular Therapeutics

Although the CD20-targeted monoclonal antibody rituximab (RTX) has revolutionized the therapeutic landscape for B-cell malignancy, relapsed and refractory disease due to RTX resistance continue to constitute major challenges, illustrating the need for better therapies. Here, we apply drug-free macromolecular therapeutics (DFMT) that amplifies CD20 cross-linking to enhance apoptosis in RTX-resistant cells. Bispecific engager (anti-CD20 Fab′ conjugated with oligonucleotide1) pretargets CD20 and the deletion of Fc-region minimizes its premature endocytosis in resistant cells that rapidly internalize and consume CD20/RTX complexes. Second-step delivery of multivalent polymeric effector (linear copolymer conjugated with multiple copies of complementary oligonucleotide 2) simultaneously hybridizes multiple CD20-bound engagers and strengthens CD20 ligation. Moreover, the restoration of CD20 expression by the pretreatment of cells with a polymer-gemcitabine conjugate, a CD20 expression enhancer, unleashes the full potential of DFMT in the CD20-deficient resistant cells. Hence, amplification of CD20 cross-linking is achieved by (1) the enhancement of surface CD20 accessibility, (2) the increase in CD20 expression, and (3) multimeric CD20 binding, which ultimately translates into the amplified activation of a wide range of innate apoptotic responses. We demonstrated that the altered molecular signaling pathway that originally results in RTX resistance could be circumvented and compensated by other DFMT-augmented pathways. Of note, our preliminary data provide proof-of-concept that CD20 cross-linking amplification emerges as an important strategy for overcoming RTX resistance

Dual Stimuli-Responsive Hybrid Polymeric Nanoparticles Self-Assembled from POSS-Based Starlike Copolymer-Drug Conjugates for Efficient Intracellular Delivery of Hydrophobic Drugs

To further fine tune drug release and enhance therapeutic effects of polyhedral oligomericsilsesquioxane (POSS)-based nanomedicine, a starlike organic–inorganic conjugate was synthesized by grafting semitelechelic <i>N</i>-(2-hydroxypropyl) methacrylamide (HPMA) copolymers to a POSS rigid core through reductively degradable disulfide bonds. The hydrophobic docetaxel (DTX) was attached to the grafts by pH-sensitive hydrazone bonds and also encapsulated into the POSS core (SP-DTX). Thus, the final amphiphilic star-shaped conjugates could self-assemble into nanoparticles and exhibited conspicuous drug-loading capacity (20.1 wt %) based on the covalently conjugated accompanied by physically encapsulated DTX. The stimuli-responsive DTX release under acidic lysosomal and reducing cytoplasmic environments was verified, leading to enhanced cytotoxicity against PC-3 human prostate carcinoma cells. To evaluate the in vivo therapeutic effects of the DTX-loaded nanovehicles objectively, a stroma-rich, prostate xenograft tumor model was generated. SP-DTX displayed uniform tumor distribution and suppressed tumor growth to a more pronounced level (tumor inhibition of 78.9%) than nonredox-sensitive SP-DTX-A (67.4%), SP-DTX-C contained DTX only in the core (65.5%) or linear P-DTX (60.7%) through enhanced depletion of cancer-associated fibroblasts and induction of apoptosis. The hybrid POSS-based polymeric nanoparticles offer an efficient approach to transport hydrophobic drugs for cancer therapy

Impaired elastin deposition in E18.5 <i>Fstl1^−/−</i> lungs and D7 <i>Fstl1^−/−</i> lung allografts.

<p>Sections of fetal lung tissues and lung allografts were Weigert stained to visualize the deposition of elastic fibers. (A) E18.5 WT lung tissue, (B) E18.5 <i>Fstl1^−/−</i> lung tissue, (C) day 7 WT lung allograft, (D) day 7 <i>Fstl1^−/−</i> lung allograft. Elastic fibers were localized along the respiratory saccules and at the tip of the growing septa (arrows) in E18.5 WT lung (A) and D7 WT lung allografts (C). Elastin fibers could be barely seen in E18.5 <i>Fstl1^−/−</i> lung (B, arrowheads) and showed a disorganized deposition (D, arrowheads) in D7 <i>Fstl1^−/−</i> lung allograft when compared to their WT controls. Quantitative image analysis showed that <i>Fstl1</i> deletion reduced the elastin fiber density, expressed as a percentage of lung parenchyma, in <i>Fstl1^−/−</i> lungs and allografts. Data are shown in the top right corner of each diagram (<i>P</i><0.05). Scale bar, 50 μm. (E) Elastin production was decreased in E18.5 <i>Fstl1^−/−</i> lungs using qRT-PCR analysis (n = 6). The graph represents the mean ± SEM of three independent experiments.</p

Differentiation of AEC-I cells and blood vessel development in lung allografts.

<p>E15.5 WT lungs were diced and implanted underneath the renal capsule. Sections of fetal lung tissues and allografts at indicated time points were immunostained with T1α or Endomucin antibodies. (A) and (E) E18.5 lung tissues, (B) and (F) P4 lung tissues, (C) and (G) day 3 allografts, (D) and (H) day 7 allografts. T1α-positive AEC-I cells were found lining the saccular lumen of fetal lung tissues and renal capsule allografts (A–D). Endomucin-positive endothelial cells were found next to the saccular epithelium of fetal lung tissues and renal capsule allografts (E–H). Green color: Endomucin; blue color: DAPI. Scale bar, 50 μm.</p

Morphogenesis of murine fetal distal lung explants in organ cultures.

<p>E15.5 WT lungs were diced and <i>in vitro</i> cultured. Microscopic analysis of explants at indicated time points. (A) day 1, (B) day 2 and (C) day 3. Scale bar, 100 μm.</p

Morphogenesis of murine fetal distal lung allografts underneath the renal capsule.

<p>E15.5 WT lungs were diced and implanted underneath the renal capsule. Sections of fetal lung tissues, explants and allografts at indicated time points were stained with Hematoxylin and eosin (H&E) and shown at low and high (inserts) magnification. (A) E15.5 lung tissue, (C) E18.5 lung tissue, (E) P4 lung tissue, (B) day 3 explant, (D) day 3 allograft, (F) day 7 allograft. Arrowheads indicate secondary septae (E–F). Scale bar, 100 μm.</p

Differentiation of AEC-II cells in lung allografts.

<p>E15.5 WT lungs were diced and implanted underneath the renal capsule. Sections of fetal lung tissues, allografts and explants at indicated time points were immunostained with pro SP-C antibody. (A) E18.5 lung tissue, (B) P4 lung tissue, (C) day 3 allograft, (D) day 7 allograft, (E) day 3 explant. Pro SP-C-positive AEC-II cells were found scattered in saccular structures of fetal lung tissues and renal capsule allografts (A–D), while, pro SP-C-positive AEC-II cells were found lining the distal acinar tubules in lung explants (E). Red color: pro SP-C; blue color: DAPI. Scale bar, 50 μm.</p

Tim-3 Expression Defines Regulatory T Cells in Human Tumors

<div><p>Tim-3, a member of the novel Tim (T cell immunoglobulin and mucin domain) family, has been reported to negatively regulate the immune responses against viral infection and had implications for autoimmune disease. However, the nature and role of Tim-3⁺ CD4 T cells in human tumors remain largely unknown. In the present study, we characterized Tim-3⁺ CD4 T cells in 100 specimens from human hepatocellular, cervical, colorectal and ovarian carcinoma patients. Compared with peripheral blood and nontumor-infiltrating lymphocytes, the lymphocytes isolated from the corresponding tumor tissues of hepatocellular, cervical, colorectal and ovarian carcinoma patients contained significantly greater proportion of Tim-3⁺ CD4 T cells. The majority of tumor-derived Tim-3⁺ CD4 T cells exhibited an impaired capacity to produce IFN-γ and IL-2, but expressed higher levels of CD25, Foxp3, CTLA-4 and GITR than their Tim-3⁻ CD4 T cell counterparts. In contrast, most Tim-3⁺ CD4 T cells isolated from the paired nontumor tissues and peripheral blood did not express these molecules. Moreover, tumor-derived Tim-3⁺ CD4 T cells, but not tumor-derived Tim-3⁻ CD4 T cells, significantly suppressed the proliferation of autologous CD8⁺ T cells <i>in vitro</i>. Notably, multi-color immunofluorescence and confocal microscopy demonstrated that Tim-3⁺Foxp3⁺CD4⁺ cells were preferentially distributed in the tumor nest rather than the peritumoral stroma of hepatocellular carcinoma. Together, our data indicate that Tim-3-expressing CD4 T cells in human tumors could represent the functional regulatory T cells which contribute to the formation of the immune-suppressive tumor micromilieu.</p> </div