Enhanced removal performance of magnesium-modified biochar for cadmium in wastewaters: Role of active functional groups, processes, and mechanisms.

In this study, a series of biochar products with different active functional groups were developed by one-pot coprecipitation method, including magnesium-modified biochar (MgBC) and functional group-grafted MgBC (Cys@MgBC, Try@MgBC, and Glu@MgBC), for effective adsorption of cadmium (Cd(II)) from wastewaters. These biochars exhibited excellent removal performance for Cd(II), particularly Cys@MgBC, whose maximum Cd(II) adsorption capacity reached 223.7 mg g-1. The highly active and weakly crystalline Mg could adsorb Cd(II) through precipitation and ion exchange, which was further promoted by the introduced functional groups through complexation and precipitation. After 120 d of natural process, the immobilization efficiency of Cd(II) by Cys@MgBC, Try@MgBC, and Glu@MgBC was still maintained at 98.7%, 95.2%, and 82.7% respectively. This study proposes and clarifies the complexation mechanism of functional group-grafted Mg-modified biochar for heavy metals, providing new insights into the practical application of these biochars.

Therapeutic adenine base editing of human hematopoietic stem cells.

In ß-thalassemia, either γ-globin induction to form fetal hemoglobin (α2γ2) or ß-globin repair to restore adult hemoglobin (α2ß2) could be therapeutic. ABE8e, a recently evolved adenine base editor variant, can achieve efficient adenine conversion, yet its application in patient-derived hematopoietic stem cells needs further exploration. Here, we purified ABE8e for ribonucleoprotein electroporation of ß-thalassemia patient CD34+ hematopoietic stem and progenitor cells to introduce nucleotide substitutions that upregulate γ-globin expression in the BCL11A enhancer or in the HBG promoter. We observed highly efficient on-target adenine base edits at these two regulatory regions, resulting in robust γ-globin induction. Moreover, we developed ABE8e-SpRY, a near-PAMless ABE variant, and successfully applied ABE8e-SpRY RNP to directly correct HbE and IVS II-654 mutations in patient-derived CD34+ HSPCs. Finally, durable therapeutic editing was produced in self-renewing repopulating human HSCs as assayed in primary and secondary recipients. Together, these results support the potential of ABE-mediated base editing in HSCs to treat inherited monogenic blood disorders.

The advanced lung cancer inflammation index predicts chemotherapy response and infection risk in multiple myeloma patients receiving induction chemotherapy.

Objective: This study aims to determine the clinical significance of the advanced lung cancer inflammation index (ALI) in predicting prognosis, chemotherapy response, and infection risk in newly diagnosed multiple myeloma (MM) patients receiving induction therapy. Methods: A retrospective analysis of the clinical characteristics and laboratory data of 111 newly diagnosed MM patients from the Haematology Department of the Third Xiangya Hospital of Central South University from January 2014 to March 2020 was performed. We first determined the relationship between ALI and overall survival (OS), as well as clinical and laboratory parameters. Second, predictive factors for chemotherapy response were analysed by univariate and multivariate regression analyses. Third, univariate regression analysis of risk factors was performed using infection as the evaluable outcome. Results: Of the 111 evaluable patients, the low ALI group (<32.7) exhibited significantly poorer survival than the high ALI group (51 months versus 77 months). Multivariable analysis showed that advanced age, chemotherapy response and serum calcium level were independent prognostic factors for OS. Better chemotherapy efficacy in the high ALI group (89.3%) than in the low ALI group (42.2%) (p < 0.001) was noted. Multivariate analysis suggested that only ALI [HR: 0.110, 95% CI (0.035-0.350), p = 0.000] is an independent predictive factor in evaluating the efficiency of induction chemotherapy. Forty patients (36.04%) presented with infection after induction chemotherapy. Univariate analysis suggested that low ALI and abnormal renal function increase risk of infection in newly diagnosed MM patients. Conclusion: Our study confirmed that ALI is not only a prognostic biomarker for newly diagnosed patients, but also predicts chemotherapy efficacy in newly diagnosed MM patients receiving induction therapy.

CRISPR-Cas9-mediated gene editing of the BCL11A enhancer for pediatric ß0/ß0 transfusion-dependent ß-thalassemia.

Gene editing to disrupt the GATA1-binding site at the +58 BCL11A erythroid enhancer could induce γ-globin expression, which is a promising therapeutic strategy to alleviate ß-hemoglobinopathy caused by HBB gene mutation. In the present study, we report the preliminary results of an ongoing phase 1/2 trial (NCT04211480) evaluating safety and efficacy of gene editing therapy in children with blood transfusion-dependent ß-thalassemia (TDT). We transplanted BCL11A enhancer-edited, autologous, hematopoietic stem and progenitor cells into two children, one carrying the ß0/ß0 genotype, classified as the most severe type of TDT. Primary endpoints included engraftment, overall survival and incidence of adverse events (AEs). Both patients were clinically well with multilineage engraftment, and all AEs to date were considered unrelated to gene editing and resolved after treatment. Secondary endpoints included achieving transfusion independence, editing rate in bone marrow cells and change in hemoglobin (Hb) concentration. Both patients achieved transfusion independence for >18 months after treatment, and their Hb increased from 8.2 and 10.8 g dl-1 at screening to 15.0 and 14.0 g dl-1 at the last visit, respectively, with 85.46% and 89.48% editing persistence in bone marrow cells. Exploratory analysis of single-cell transcriptome and indel patterns in edited peripheral blood mononuclear cells showed no notable side effects of the therapy.

Wenxin Granule Ameliorates Hypoxia/Reoxygenation-Induced Oxidative Stress in Mitochondria via the PKC-δ/NOX2/ROS Pathway in H9c2 Cells.

Myocardial infarction and following reperfusion therapy-induced myocardial ischemia/reperfusion (I/R) injury have been recognized as an important subject of cardiovascular disease with high mortality. As the antiarrhythmic agent, Wenxin Granule (WXG) is widely used to arrhythmia and heart failure. In our pilot study, we found the antioxidative potential of WXG in the treatment of myocardial I/R. This study is aimed at investigating whether WXG could treat cardiomyocyte hypoxia/reoxygenation (H/R) injury by inhibiting oxidative stress in mitochondria. The H9c2 cardiomyocyte cell line was subject to H/R stimuli to mimic I/R injury in vitro. WXG was added to the culture medium 24 h before H/R exposing as pretreatment. Protein kinase C-δ (PKC-δ) inhibitor rottlerin or PKC-δ lentivirus vectors were conducted on H9c2 cells to downregulate or overexpress PKC-δ protein. Then, the cell viability, oxidative stress levels, intracellular and mitochondrial ROS levels, mitochondrial function, and apoptosis index were analyzed. In addition, PKC-δ protein expression in each group was verified by western blot analysis. Compared with the control group, the PKC-δ protein level was significantly increased in the H/R group, which was remarkably improved by WXG or rottlerin. PKC-δ lentivirus vector-mediated PKC-δ overexpression was not reduced by WXG. WXG significantly improved H/R-induced cell injury, lower levels of SOD and GSH/GSSG ratio, higher levels of MDA, intracellular and mitochondrial ROS content, mitochondrial membrane potential and ATP loss, mitochondrial permeability transition pore opening, NOX2 activation, cytochrome C release, Bax/Bcl-2 ratio and cleaved caspase-3 increasing, and cell apoptosis. Similar findings were obtained from rottlerin treatment. However, the protective effects of WXG were abolished by PKC-δ overexpression, indicating that PKC-δ was a potential target of WXG treatment. Our findings demonstrated a novel mechanism by which WXG attenuated oxidative stress and mitochondrial dysfunction of H9c2 cells induced by H/R stimulation via inhibitory regulation of PKC-δ/NOX2/ROS signaling.

Cyclic Tripeptide-based Potent and Selective Human SIRT5 Inhibitors.

BACKGROUND: SIRT5 is one of the seven members (SIRT1-7) of the mammalian sirtuin family of protein acyl-lysine deacylase enzymes. In recent years, important regulatory roles of SIRT5 in (patho)physiological conditions (e.g. metabolism and cancer) have been increasingly demonstrated. For a better biological understanding and therapeutic exploitation of the SIRT5- catalyzed deacylation reaction, more effort on identifying potent and selective SIRT5 inhibitors beyond those currently known would be rewarding. OBJECTIVE: In the current study, we would like to see if it would be possible to develop potent and selective SIRT5 inhibitory lead compounds with a novel structural scaffold than those of the currently known potent and selective SIRT5 inhibitors. METHODS: In the current study, six N-terminus-to-side chain cyclic tripeptides (i.e. 8-13) each harboring the thiourea-type catalytic mechanism-based SIRT5 inhibitory warhead Nε-carboxyethylthiocarbamoyl- lysine as the central residue were designed, synthesized by the Nα-9- fluorenylmethoxycarbonyl (Fmoc) chemistry-based solid phase peptide synthesis (SPPS) on the Rink amide 4-methylbenzhydrylamine (MBHA) resin, purified by the semi-preparative reversedphase high performance liquid chromatography (RP-HPLC), characterized by the high-resolution mass spectrometry (HRMS); and were evaluated by the in vitro sirtuin inhibition assay and the in vitro proteolysis assay. RESULTS: Among the cyclic tripeptides 8-13, we found that 10 exhibited a potent (IC50 ~2.2 µM) and selective (≥60-fold over the SIRT1/2/3/6-catalyzed deacylation reactions) inhibition against the SIRT5-catalyzed desuccinylation reaction. Moreover, 10 was found to exhibit a ~42.3-fold stronger SIRT5 inhibition and a greater proteolytic stability than its linear counterpart 14. CONCLUSION: With a novel and modular structural scaffold as compared with those of all the currently reported potent and selective SIRT5 inhibitors, 10 could be also a useful and feasible lead compound for the quest for superior SIRT5 inhibitors as potential chemical/pharmacological probes of SIRT5 and therapeutics for human diseases in which SIRT5 desuccinylase activity is upregulated.

Formin mDia1 contributes to migration and epithelial-mesenchymal transition of tubular epithelial cells exposed to TGF-ß1.

Renal tubular epithelial cells may undergo epithelial-mesenchymal transition (EMT) in response to stimuli, such as transforming growth factor (TGF)-ß1, leading to myofibroblast activation and renal fibrosis. The formin mDia1 is required for nucleation and polymerization of actin and the microtubule cytoskeleton. The present study sought to explore the role of mDia1 in EMT of tubular epithelial cells. A rat model of unilateral ureteral obstruction (UUO) was established. The expression of TGF-ß1, collagen I, collagen III, and mDia1 in the kidneys was examined at day 7 after surgery. The effect of mDia1 on EMT was explored in NRK-52E cells by exposing them to TGF-ß1. Increased expression of TGF-ß1, collagen I, collagen III, and mDia1 was found in obstructive kidneys of UUO model rats. Exposing rat tubular epithelial cells to TGF-ß1 promoted collagen I and collagen III expression but had no effect on mDia1 expression. Silencing mDia1 expression impeded epithelial cell migration as well as reduced TGF-ß1, collagen, and Profilin1 expression, whereas mDia1 overexpression exerted an opposite effect. Furthermore, mDia1 regulated the expression of vimentin, α-smooth muscle actin, and E-cadherin and focal adhesion-kinase (FAK)/Src activation through Profilin1. Inhibition of the mDia1 activator RhoA by fasudil reversed EMT, and FAK/Src activation induced by mDia1. In conclusion, mDia1 regulated tubular epithelial cell migration, collagen expression, and EMT in NRK-52E cells exposed to TGF-ß1. Thus, suppression of mDia1 activation might be a strategy to counteract renal fibrosis.

Development of Stabilized Peptide-Based PROTACs against Estrogen Receptor α.

Peptide modulators targeting protein-protein interactions (PPIs) exhibit greater potential than small-molecule drugs in several important aspects including facile modification and relative large contact surface area. Stabilized peptides constructed by variable chemistry methods exhibit improved peptide stability and cell permeability compared to that of the linears. Herein, we designed a stabilized peptide-based proteolysis-targeting chimera (PROTAC) targeting estrogen receptor α (ERα) by tethering an N-terminal aspartic acid cross-linked stabilized peptide ERα modulator (TD-PERM) with a pentapeptide that binds the Von Hippel-Lindau (VHL) E3 ubiquitin ligase complex. The resulting heterobifunctional peptide (TD-PROTAC) selectively recruits ERα to the VHL E3 ligase complex, leading to the degradation of ERα in a proteasome-dependent manner. Compared with the control peptides, TD-PROTAC shows significantly enhanced activities in reducing the transcription of the ERα-downstream genes and inhibiting the proliferation of ERα-positive breast cancer cells. In addition, in vivo experiments indicate that TD-PROTAC leads to tumor regression in the MCF-7 mouse xenograft model. This work is a successful attempt to construct PROTACs based on cell-permeable stabilized peptides, which significantly broadens the chemical space of PROTACs and stabilized peptides.

The development of activatable lytic peptides for targeting triple negative breast cancer.

Cytolytic peptides are an emerging class of promising cancer therapeutics shown to overcome drug resistance. They eliminate cancer cells via disruption of the phospholipid bilayer of cell membranes, a mechanism that differentiates it from traditional treatments. However, applications of lytic peptides via systematic administration are hampered by nonspecific toxicity. Here, we describe activatable, masked lytic peptides that are conjugated with anionic peptides via a cleavable linker sensitive to matrix metalloproteinases (Ac-w-ßA-e8-XPLG*LAG-klUklUkklUklUk-NH2; lower case letters in the sequences represent D-amino-acids, U=Aib, α-aminoisobutyric acid, *cleavage site). The peptides were activated upon being introduced into the triple negative breast cancer cell line MDA-MB-231, which overexpresses secreted matrix metalloproteinases, to selectively cleave the peptide linker. Our results indicate that the activatable design could be applied to improve the targeting ability of lytic peptides.

Structural Basis of Inhibition of ERα-Coactivator Interaction by High-Affinity N-Terminus Isoaspartic Acid Tethered Helical Peptides.

Direct inhibition of the protein-protein interaction of ERα and its endogenous coactivators with a cell permeable stabilized peptide may offer a novel, promising strategy for combating ERα positive breast cancers. Here, we report the co-crystal structure of a helical peptide stabilized by a N-terminal unnatural cross-linked aspartic acid (TD) in complex with the ERα ligand binding domain (LBD). We designed a series of peptides and peptide 6 that showed direct and high-affinity binding to ERα with selective antiproliferative activity in ERα positive breast cancer cells. The co-crystal structure of the TD-stabilized peptide 6 in complex with ERα LBD further demonstrates that it forms an α helical conformation and directly binds at the coactivator binding site of ERα. Further studies showed that peptide 6W could potently inhibit cellular ERα's transcriptional activity. This approach demonstrates the potential of TD stabilized peptides to modulate various intracellular protein-protein interactions involved in a range of disorders.

Effect of Stapling Architecture on Physiochemical Properties and Cell Permeability of Stapled α-Helical Peptides: A Comparative Study.

Stapled peptides have emerged as a new class of targeting molecules with high binding affinity and specificity for intracellular undruggable targets. Their ability to penetrate cell membranes is exceptionally intriguing but remains elusively and controversially discussed. To understand the effect of stapling architectures on their physiochemical properties and to aid in promoting their cell permeability, we report herein a comparative study on the physiochemical properties and cell permeability of stapled α-helical peptides with different types of crosslinks. We highlight the decisive impact of the intrinsic properties of the crosslinks on cell permeability rather than the helical contents of the peptides in model amphipathic sequences targeting estrogen receptor-coactivator interaction. We envision this finding to shed further light on the chemical optimization of stapled α-helical peptides or macrocyclic cell-penetrating peptides for enhanced cell penetration.

Sirtuin Inhibition: Strategies, Inhibitors, and Therapeutic Potential.

The ß-NAD+-dependent Nε-acyl-lysine deacylation reaction catalyzed by sirtuin family members has been increasingly demonstrated to be important in regulating multiple crucial cellular processes and has also been proposed to be a therapeutic target for multiple human diseases. Accordingly, its inhibitors have been actively pursued over the past few years. In addition, we have also seen the pharmacological assessment of sirtuin inhibitory compounds, although to a lesser extent. In this review, we first discuss how sirtuin inhibitors were discovered with the use of various approaches. We then follow with a discussion of pharmacological studies using sirtuin inhibitors. Our aim here is to set a stage for developing future superior sirtuin inhibitors and for an expanded effort in exploiting inhibitors to explore and/or validate the therapeutic potential stemming from the inhibition of the sirtuin-catalyzed deacylation reaction.

Improving cell penetration of helical peptides stabilized by N-terminal crosslinked aspartic acids.

Cell penetration and nucleus translocation efficiency are important for the cellular activities of peptide therapeutics. For helical peptides stabilized by N-terminal crosslinked aspartic acid, correlations between their penetration efficiency/nucleus translocation and physicochemical properties were studied. An increase in hydrophobicity and isoelectric point will promote cellular uptake and nucleus translocation of stabilized helices.

Crosslinked Aspartic Acids as Helix-Nucleating Templates.

Described is a facile helix-nucleating template based on a tethered aspartic acid at the N-terminus [terminal aspartic acid (TD)]. The nucleating effect of the template is subtly influenced by the substituent at the end of the side-chain-end tether as indicated by circular dichroism, nuclear magnetic resonance, and molecular dynamics simulations. Unlike most nucleating strategies, the N-terminal amine is preserved, thus enabling further modification. Peptidomimetic estrogen receptor modulators (PERMs) constructed using this strategy show improved therapeutic properties. The current strategy can be regarded as a good complement to existing helix-stabilizing methods.