Efficient Capture and Release of the Rare-Earth Element Neodymium in Aqueous Solution by Recyclable Covalent Organic Frameworks.

Rare-earth elements (REEs) are present in a broad range of critical materials. The development of solid adsorbents for REE capture could enable the cost-effective recycling of REE-containing magnets and electronics. In this context, covalent organic frameworks (COFs) are promising candidates for REE adsorption due to their exceptionally high surface area. Despite having attractive physical properties, COFs are heavily underutilized for REE capture applications due to their limited lifecycle in aqueous acidic environments, as well as synthetic challenges associated with the incorporation of ligands suitable for REE capture. Here, we show how the Ugi multicomponent reaction can be leveraged to postsynthetically modify imine-based COFs for the introduction of a diglycolic acid (DGA) moiety, an efficient scaffold for REE capture. The adsorption capacity of the DGA-functionalized COF was found to be more than 40 times higher than that of the pristine imine COF precursor and more than four times higher than that of the next-best reported DGA-functionalized solid support. This rationally designed COF has appealing characteristics of high adsorption capacity, fast and efficient capture and release of the REE ions, and reliable recyclability, making it one of the most promising adsorbents for solid-liquid REE ion extractions reported to date.

Allosteric nanobodies to study the interactions between SOS1 and RAS.

Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1•RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.

New Perspectives in Colorectal Cancers Treatment, the Role of MicroRNAs.

The main epidemiological and clinical data on colorectal cancer, as well as the features of molecular pathology, are discussed in the literature review. Efforts are being putto identify promising targets, particularly small non-coding nucleotide sequences, which can lead to new treatments for this disease. The discovery of significant mutations that contribute to the development of colorectal tumors is a major step in the advancement of molecular oncology, as these mutations give rise to heterogeneous tumors that differ in their origin. These mutations play a significant role in the progression of the disease and are now being targeted for treatment. The prognosis for a disease is influenced by the patient's sensitivity to antitumor therapy. However, new approaches to finding effective targets for antitumor treatments face new fundamental challenges due to clinical issues. These issues include the epigenetic regulation of markers of oncogenesis, which allows for the development of new therapeutic strategies. RNA interference, in particular, has been linked to non-copying RNA sequences such as microRNAs. These microRNAs are associated with certain processes that can influence all aspects of oncogenesis. The diversity of microRNAs allows for a differentiated approach when treating tumors in various locations.

Multiple Administration of Dexamethasone Possesses a Deferred Long-Term Effect to Glycosylated Components of Mouse Brain.

Glucocorticoids are used during glioblastoma treatment to prevent the cerebral edema effect surrounding normal brain tissue. The aim of our study was to investigate the long-term effects of multiple administrations of glucocorticoids onto the glycosylated components (proteoglycans and glycosaminoglycans) of normal brain extracellular matrix and the glucocorticoid receptor (GR, Nr3c1) in an experimental model in vivo. Two-month-old male C57Bl/6 mice (n = 90) were injected intraperitoneally with various doses of dexamethasone (DXM) (1; 2.5 mg/kg) for 10 days. The mRNA levels of the GR, proteoglycans core proteins, and heparan sulfate metabolism-involved genes were determined at the 15th, 30th, 60th, and 90th days by a real-time RT-PCR. The glycosaminoglycans content was studied using dot blot and staining with Alcian blue. A DXM treatment increased total GAG content (2-fold), whereas the content of highly sulfated glycosaminoglycans decreased (1.5-2-fold). The mRNA level of the heparan sulfate metabolism-involved gene Hs3St2 increased 5-fold, the mRNA level of Hs6St2 increased6-7-fold, and the mRNA level of proteoglycan aggrecan increased 2-fold. A correlation analysis revealed an association between the mRNA level of the GR and the mRNA level of 8 of the 14 proteoglycans-coding and 4 of the 13 heparan sulfate metabolism-involved genes supporting GR involvement in the DXM regulation of the expression of these genes. In summary, multiple DXM administrations led to an increase in the total GAG content and reorganized the brain extracellular matrix in terms of its glycosylation pattern.

Enantioselective, BroÌ·nsted Acid-Catalyzed Anti-selective Hydroamination of Alkenes.

Chiral pyrrolidines are common structural motives in natural products as well as active pharmaceutical ingredients, explaining the need for methods for their enantioselective synthesis. While several, often metal-catalyzed, methods for their preparation do exist, the enantioselective synthesis of pyrrolidines containing quaternary stereocenters remains challenging. Herein, we report a BroÌ·nsted acid-catalyzed intramolecular hydroamination that provides such pyrrolidines from simple starting materials in high yield and enantioselectivity. Key to an efficient reaction was the use of an electron-deficient protective group on nitrogen, the common nosyl-protecting group, to avoid deactivation of the BroÌ·nsted acid by deprotonation. The reaction proceeds as a stereospecific anti-addition indicating a concerted reaction. Furthermore, kinetic studies show Michaelis-Menten behavior, suggesting the formation of a precomplex similar to those observed in enzymatic catalysis.

Beyond the VSG layer: Exploring the role of intrinsic disorder in the invariant surface glycoproteins of African trypanosomes.

In the bloodstream of mammalian hosts, African trypanosomes face the challenge of protecting their invariant surface receptors from immune detection. This crucial role is fulfilled by a dense, glycosylated protein layer composed of variant surface glycoproteins (VSGs), which undergo antigenic variation and provide a physical barrier that shields the underlying invariant surface glycoproteins (ISGs). The protective shield's limited permeability comes at the cost of restricted access to the extracellular host environment, raising questions regarding the specific function of the ISG repertoire. In this study, we employ an integrative structural biology approach to show that intrinsically disordered membrane-proximal regions are a common feature of members of the ISG super-family, conferring the ability to switch between compact and elongated conformers. While the folded, membrane-distal ectodomain is buried within the VSG layer for compact conformers, their elongated counterparts would enable the extension beyond it. This dynamic behavior enables ISGs to maintain a low immunogenic footprint while still allowing them to engage with the host environment when necessary. Our findings add further evidence to a dynamic molecular organization of trypanosome surface antigens wherein intrinsic disorder underpins the characteristics of a highly flexible ISG proteome to circumvent the constraints imposed by the VSG coat.

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae.

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while "hovering" over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

Amyloid Typing in Cardiac Amyloidosis Using Western Blotting.

BACKGROUND: Cardiac amyloidosis (CA) is characterized by the extracellular deposition of misfolded protein in the heart. Precise identification of the amyloid type is often challenging, but critical, since the treatment and prognosis depend on the disease form and the type of deposited amyloid. Coexistence of clinical conditions such as old age, monoclonal gammopathy, chronic inflammation, or peripheral neuropathy in a patient with cardiomyopathy creates a differential diagnosis between the major types of CA: amyloidosis light chains (AL), amyloidosis transthyretin (ATTR) and amyloidosis A (AA). OBJECTIVES: To demonstrate the utility of the Western blotting (WB)-based amyloid typing method in patients diagnosed with cardiac amyloidosis where the type of amyloid was not obvious based on the clinical context. METHODS: Congo red positive endomyocardial biopsy specimens were studied in patients where the type of amyloid was uncertain. Amyloid proteins were extracted and identified by WB. Mass spectrometry (MS) of the electrophoretically resolved protein-in-gel bands was used for confirmation of WB data. RESULTS: WB analysis allowed differentiation between AL, AA, and ATTR in cardiac biopsies based on specific immunoreactivity of the electrophoretically separated proteins and their characteristic molecular weight. The obtained results were confirmed by MS. CONCLUSIONS: WB-based amyloid typing method is cheaper and more readily available than the complex and expensive gold standard techniques such as MS analysis or immunoelectron microscopy. Notably, it is more sensitive and specific than the commonly used immunohistochemical techniques and may provide an accessible diagnostic service to patients with amyloidosis in Israel.

Retroperitoneal Liposarcoma: Rational Extent of Surgery Tailored to Grade of Malignancy.

This study was aimed at determining the indications for combined and organ-preserving operations. The study included 190 patients with retroperitoneal liposarcoma (RLPS). The influence of the following factors on the overall survival (OS) and recurrence-free survival (RFS) were studied: involvement of adjacent organs in the tumor, volume of surgical intervention. OS and RFS were worse in pathologically confirmed visceral invasion in the both RLPS low grade and high grade (p = 0.000). In RLPS low grade, there was no significant difference in OS and RFS between the group of patients who underwent combined surgery without confirmed visceral invasion and the group of patients who underwent organ-preserving surgery (p > 0.080). In RLPS high grade, OS and RFS were higher in the group of patients who underwent combined surgery without confirmed visceral invasion than in the group of patients who underwent organ-preserving surgery (p < 0.050). In RLPS low grade, it is advisable to perform organ-preserving operations, including nephrosaving operations. In RLPS high grade, the organ-preserving operations worsen long-term results and prognosis. Combined operations including nephrectomy are justified in RLPS high grade.

Prediction of Recurrence-Free and Overall Survival in Retroperitoneal Well-Differentiated and Dedifferentiated Liposarcoma After Surgery.

This study was aimed at creating an effective model for predicting the course of the disease in retroperitoneal well-differentiated (WDLPS) and dedifferentiated (DDLPS) liposarcomas after surgery. The study included 111 patients with WDLPS and 74 patients with DDLPS. We developed a methodology for stratification of patients into prognostic groups. Overall survival (OS) and recurrence-free survival (RFS) were analyzed in accordance with it. The highest OS was achieved in the group "favorable prognosis," while the shortest OS was in the group "extremely poor prognosis" (p < 0.001). The median OS in the "favorable prognosis" group was 225 (95% CI, 174, 276) months; "intermediate prognosis" - 130 (95% CI, 115, 145) months; "poor prognosis" - 90 (95% CI, 79, 101) months; and "extremely poor prognosis" - 22 (95% CI, 15, 29) months. The highest RFS was achieved in the group "favorable prognosis," while the shortest RFS was achieved in the group "extremely poor prognosis" (p < 0.001). The median RFS in the "favorable prognosis" group was 80 (95% CI, 65, 95) months; "intermediate prognosis" - 47 (95% CI, 33, 61) months; "poor prognosis" - 26 (95% CI, 24, 28) months; "extremely poor prognosis" - 10 (95% CI, 6, 14) months. The method of predicting recurrence-free and overall survival demonstrates an adequate distribution of patients and the reliability of intergroup differences in the survival rate.

Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Toxin:antitoxin ratio sensing autoregulation of the Vibrio cholerae parDE2 module.

The parDE family of toxin-antitoxin (TA) operons is ubiquitous in bacterial genomes and, in Vibrio cholerae, is an essential component to maintain the presence of chromosome II. Here, we show that transcription of the V. cholerae parDE2 (VcparDE) operon is regulated in a toxin:antitoxin ratio-dependent manner using a molecular mechanism distinct from other type II TA systems. The repressor of the operon is identified as an assembly with a 6:2 stoichiometry with three interacting ParD2 dimers bridged by two ParE2 monomers. This assembly docks to a three-site operator containing 5'- GGTA-3' motifs. Saturation of this TA complex with ParE2 toxin results in disruption of the interface between ParD2 dimers and the formation of a TA complex of 2:2 stoichiometry. The latter is operator binding-incompetent as it is incompatible with the required spacing of the ParD2 dimers on the operator.

Biophysical characterization of the Plasmodium falciparum circumsporozoite protein's N-terminal domain.

The circumsporozoite protein (CSP) is the main surface antigen of the Plasmodium sporozoite (SPZ) and forms the basis of the currently only licensed anti-malarial vaccine (RTS,S/AS01). CSP uniformly coats the SPZ and plays a pivotal role in its immunobiology, in both the insect and the vertebrate hosts. Although CSP's N-terminal domain (CSPN ) has been reported to play an important role in multiple CSP functions, a thorough biophysical and structural characterization of CSPN is currently lacking. Here, we present an alternative method for the recombinant production and purification of CSPN from Plasmodium falciparum (PfCSPN ), which provides pure, high-quality protein preparations with high yields. Through an interdisciplinary approach combining in-solution experimental methods and in silico analyses, we provide strong evidence that PfCSPN is an intrinsically disordered region displaying some degree of compaction.

Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called "ascorbate peroxidase-related" (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from Chlamydomonas reinhardtii referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arginine transport (TAT) motif for lumen translocation and the metal-binding MxxM motif. We also successfully expressed recombinant APX2 in Escherichia coli. Our in vitro results showed that the peroxidase activity of APX2 was detected with guaiacol but not with ascorbate as an electron donor. Furthermore, APX2 can bind both copper and heme, as evidenced by spectroscopic, and fluorescence experiments. These findings suggest a potential interaction between APX2 and plastocyanin, the primary copper-containing enzyme within the thylakoid lumen of the chloroplasts. Predictions from structural models and evidence from 1H-NMR experiments suggest a potential interaction between APX2 and plastocyanin, emphasizing the influence of APX2 on the copper-binding abilities of plastocyanin. In summary, our results propose a significant role for APX2 as a regulator in copper transfer to plastocyanin. This study sheds light on the unique properties of APX-R enzymes and their potential contributions to the complex processes of photosynthesis in green algae.

Safety and Efficacy of Neoadjuvant Chemoimmunotherapy in Gastric Cancer Patients with a PD-L1 Positive Status: A Case Report.

INTRODUCTION: The landscape of gastric cancer treatment has changed owing to the widespread use of immune checkpoint inhibitors. Autophagy, involved in regulating the immune system, is a potential trigger of immunity in tumors. This study aims to find molecular-based evidence for the effectiveness of FLOT chemotherapy with immune checkpoint inhibitors in gastric cancer patients. MATERIALS AND METHODS: Three patients with advanced gastric cancer received FLOT neoadjuvant chemotherapy with immunotherapy and surgery. IHC was used to determine the PD-L1 status. Real-time PCR was used to analyze expression patterns of transcriptional growth factors, AKT/mTOR signaling components, PD-1, PD-L1, PD-L2 and LC3B. The LC3B content was measured via Western blotting analysis. RESULTS: The combination of FLOT neoadjuvant chemotherapy and immunotherapy was found to be efficient in patients with a PD-L1-positive status. Gastric tumors with a PD-L1-positive status exhibited autophagy activation and decreased PD-1 expression. CONCLUSIONS: FLOT chemotherapy combined with immune checkpoint inhibitors showed high efficacy in gastric cancer patients with a positive PD-L1 status. Autophagy was involved in activating the tumor immunity. Further research is needed to clarify the mechanism of effective anticancer treatment.

Identification of small molecule antivirals against HTLV-1 by targeting the hDLG1-Tax-1 protein-protein interaction.

Human T-cell leukemia virus type-1 (HTLV-1) is the first pathogenic retrovirus discovered in human. Although HTLV-1-induced diseases are well-characterized and linked to the encoded Tax-1 oncoprotein, there is currently no strategy to target Tax-1 functions with small molecules. Here, we analyzed the binding of Tax-1 to the human homolog of the drosophila discs large tumor suppressor (hDLG1/SAP97), a multi-domain scaffolding protein involved in Tax-1-transformation ability. We have solved the structures of the PDZ binding motif (PBM) of Tax-1 in complex with the PDZ1 and PDZ2 domains of hDLG1 and assessed the binding of 10 million molecules by virtual screening. Among the 19 experimentally confirmed compounds, one systematically inhibited the Tax-1-hDLG1 interaction in different biophysical and cellular assays, as well as HTLV-1 cell-to-cell transmission in a T-cell model. Thus, our work demonstrates that interactions involving Tax-1 PDZ-domains are amenable to small-molecule inhibition, which provides a framework for the design of targeted therapies for HTLV-1-induced diseases.

Evolutionary conservation of the structure and function of meiotic Rec114-Mei4 and Mer2 complexes.

Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114-Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.

Functional Activity of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes on a Mouse Renal Subcapsular Xenograft Model.

In the treatment of coronary heart disease, the most promising approach for replacing lost contractile elements involves obtaining cardiomyocytes through cardiac differentiation of pluripotent cells. The objective of this study is to develop a technology for creating a functional layer of cardiomyocytes derived from iPSCs, capable of generating rhythmic activity and synchronous contractions. To expedite the maturation of cardiomyocytes, a renal subcapsular transplantation model was employed in SCID mice. Following explantation, the formation of the cardiomyocyte contractile apparatus was assessed using fluorescence and electron microscopy, while the cytoplasmic oscillation of calcium ions was evaluated through visualization using the fluorescent calcium binding dye Fluo-8. The results demonstrate that transplanted human iPSC-derived cardiomyocyte cell layers, placed under the fibrous capsules of SCID mouse kidneys (for up to 6 weeks), initiate the development of an organized contractile apparatus and retain functional activity along with the ability to generate calcium ion oscillations even after removal from the body.

Dexamethasone Inhibits Heparan Sulfate Biosynthetic System and Decreases Heparan Sulfate Content in Orthotopic Glioblastoma Tumors in Mice.

Glioblastoma (GB) is an aggressive cancer with a high probability of recurrence, despite active chemoradiotherapy with temozolomide (TMZ) and dexamethasone (DXM). These systemic drugs affect the glycosylated components of brain tissue involved in GB development; however, their effects on heparan sulfate (HS) remain unknown. Here, we used an animal model of GB relapse in which SCID mice first received TMZ and/or DXM (simulating postoperative treatment) with a subsequent inoculation of U87 human GB cells. Control, peritumor and U87 xenograft tissues were investigated for HS content, HS biosynthetic system and glucocorticoid receptor (GR, Nr3c1). In normal and peritumor brain tissues, TMZ/DXM administration decreased HS content (5-6-fold) but did not affect HS biosynthetic system or GR expression. However, the xenograft GB tumors grown in the pre-treated animals demonstrated a number of molecular changes, despite the fact that they were not directly exposed to TMZ/DXM. The tumors from DXM pre-treated animals possessed decreased HS content (1.5-2-fold), the inhibition of HS biosynthetic system mainly due to the -3-3.5-fold down-regulation of N-deacetylase/N-sulfotransferases (Ndst1 and Ndst2) and sulfatase 2 (Sulf2) expression and a tendency toward a decreased expression of the GRalpha but not the GRbeta isoform. The GRalpha expression levels in tumors from DXM or TMZ pre-treated mice were positively correlated with the expression of a number of HS biosynthesis-involved genes (Ext1/2, Ndst1/2, Glce, Hs2st1, Hs6st1/2), unlike tumors that have grown in intact SCID mice. The obtained data show that DXM affects HS content in mouse brain tissues, and GB xenografts grown in DXM pre-treated animals demonstrate attenuated HS biosynthesis and decreased HS content.