The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading fast worldwide. There is a pressing need to understand how the virus counteracts host innate immune responses. Deleterious clinical manifestations of coronaviruses have been associated with virus-induced direct dysregulation of innate immune responses occurring via viral macrodomains located within nonstructural protein-3 (Nsp3). However, no substantial information is available concerning the relationship of macrodomains to the unusually high pathogenicity of SARS-CoV-2. Here, we show that structural evolution of macrodomains may impart a critical role to the unique pathogenicity of SARS-CoV-2. Using sequence, structural, and phylogenetic analysis, we identify a specific set of historical substitutions that recapitulate the evolution of the macrodomains that counteract host immune response. These evolutionary substitutions may alter and reposition the secondary structural elements to create new intra-protein contacts and, thereby, may enhance the ability of SARS-CoV-2 to inhibit host immunity. Further, we find that the unusual virulence of this virus is potentially the consequence of Darwinian selection‐driven epistasis in protein evolution. Our findings warrant further characterization of macrodomain-specific evolutionary substitutions in in vitro and in vivo models to determine their inhibitory effects on the host immune system.

Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophilization-based method for isolating exosomes from cultured cells. The isolated exosomes were characterized for protein content using Bradford assay, and for size distribution and shape using scanning electron microscopy (SEM) and nanoparticles tracking analysis (NTA). In addition, CD63, CD9, CD81, HSP70 and TSG101 were evaluated as essential exosomal surface markers using Western blot. Drug loading and release studies were performed to confirm their drug delivery properties using an in vitro model. Exosomes were also loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. All these characterizations confirmed successful exosome isolation with measurements of less than 150 nm, having a typical shape, and by expressing the known exosome surface protein markers. Finally, tyrosine kinase inhibitors (dasatinib and ponatinib) were loaded on the exosomes to evaluate their anticancer effects on leukemia cells (K562 and engineered Ba/F3-BCR-ABL) using MTT and Annexin-PI assays. The expression of MUC1 protein on the exosomes isolated from MCF-7 cells also indicated that their potential diagnostic properties were intact. In conclusion, we developed a new method for exosome isolation from cultured cells. These exosomes met all the essential requirements in terms of characterization, drug loading and release ability, and inhibition of proliferation and apoptosis induction in Ph+ leukemia cells. Based on these results, we are confident in presenting the lyophilization-based exosome isolation method as an alternative to traditional techniques for exosome isolation from cultured cells.

• Synthesis of series of novel Pd(II) complexes with NNO-donor ligands. • Subtle variations of electronics and hydrophobicity on ligand arms of complexes. • Pd(II) complex with impressive inhibitory effects against breast cancer cell lines (MDA-MB-231 and MCF-7) • Cancerous cells showed Apoptotic mode of cell death. • Molecular docking studies of most active palladium complex with tyrosine kinases—an integral target in cancer research.

Ionizable lipid nanoparticles (LNPs) are widely used for delivery of CRISPR/Cas9 payloads to hepatocytes, but conventional hepatic uptake is strongly influenced by adsorption of apolipoprotein E and subsequent low-density lipoprotein receptor (LDLR)-mediated internalization. This dependence may limit specificity and reduce efficacy in LDLR-deficient settings. Here, we designed an aptamer-functionalized LNP platform to enable hepatocyte-selective genome editing through an LDLR-independent route and validated its performance using a genetically defined LDLR-knockout HepG2 model. Ionizable LNPs co-encapsulating Cas9 mRNA and an LDLR-targeting guide RNA were surface-decorated with the hepatocellular carcinoma-targeting TLS11a aptamer using thiol-maleimide chemistry. Comprehensive physicochemical analysis using cryo-electron microscopy, dynamic light scattering, pKa titration, UV and circular dichroism spectroscopy, X-ray photoelectron spectroscopy, and molecular beacon assays confirmed uniform nanoparticles of approximately 105 nm, preserved mRNA integrity, retained endosomal charge-switching behavior with a pKa of approximately 6.3 to 6.5, and maintained correctly folded surface-displayed TLS11a. TLS11a decoration increased Cas9 mRNA delivery to HepG2 cells from 39% to 79% Cy5-positive cells, while reducing uptake in receptor-low control cells, supporting aptamer-associated and cell-preferential delivery. In parallel, CRISPR/Cas9-mediated deletion of LDLR exon 2 generated a validated LDLR-deficient HepG2 line, confirmed at genomic, transcript, and protein levels. LDLR loss reduced LDL binding and uptake by approximately 85%, while transferrin uptake was preserved, indicating selective impairment of LDLR-dependent endocytosis. Cholesterol depletion activated the SCAP-SREBP-2 pathway and induced cholesterol biosynthesis genes. Together, these findings establish a modular aptamer-guided LNP system for targeted genome-editing delivery and a validated LDLR-null hepatocyte model for studying LDLR-dependent biology and disease.

Brain disorders pose a significant global healthcare challenge due to their complexity and profound impact on individuals and society. This review explores recent advancements in brain disease research, deepening our understanding and progress towards treatments. Technological innovations, like high-resolution imaging and optogenetics, play a pivotal role in illuminating cellular structures and dissecting neural circuits. Genetic analysis identifies dis-ease-associated genes and mutations, yet translating findings to clinical applications remains challenging. Epigenomics, transcriptomics, and proteomics provide molecular insights, un-covering disrupted pathways in conditions like Alzheimer's disease. Stem cell models elucidate developmental mechanisms, and immunotherapies, including CAR T cells, emerge as prom-ising strategies for brain cancers like glioblastoma. Genome editing tools, exemplified by CRISPR, enable precise modification of disease genes in models, deepening our understanding of pathogenesis. Multifaceted approaches integrating human studies, animal models, and bi-oinformatics propel the field forward. However, complexities persist, as disease specificity for cell types or brain regions remains an enigma, and genetic factors interact with environmental influences, highlighting disease intricacies. Ultimately, interdisciplinary efforts combining bi-ophysical and psychospiritual perspectives are essential to fully understand brain health and unlock transformative treatments for these debilitating disorders. A comprehensive under-standing of brain disorders is vital to improve the quality of life for millions and alleviate the global burden of neurological afflictions. Continued collaborative research will lead to an era of improved brain health worldwide.

BCL11A/EVI9, a zinc-finger protein primarily expressed in brain and hematopoietic cells, plays a central role in lymphocyte development, gamma-globin suppression, spinal neuron development, sensory innervation, neuronal polarity, migration, and is associated with microcephaly and dysregulated brain-related genes, offering therapeutic potential for sickle cell disease. The function of the transcriptional regulator is intricately linked to its structural organization, which determines its ability to interact with specific DNA sequences and modulate gene expression. BCL11A boasts multiple domains, including six C2H2 zinc fingers, a C2HC zinc finger, a NuRD-interacting domain, an acidic domain, and a proline-rich domain. In the present study, we delve into the intricate structure and function of the zinc finger domains located in the BCL11A gene, which plays a crucial role in regulating the expression of gamma-globin gene. Specifically, three C2H2-type zinc finger domains, Znf4, Znf5, and Znf6, within BCL11A, are known to bind to DNA. Znf4 and Znf5 demonstrate a significant interaction with the TGACCA motif in the gamma-globin −115 HPFH region sequence, contributing substantially to DNA binding specificity. Although Znf3 and Znf6 also interact with DNA, their contributions are comparatively minor. Employing CRISPR-Cas9 technology, targeted genomic deletions of Znf4 exhibit high efficiency, opening doors for further research. Edited CD34+ cells successfully differentiate into erythrocytes without impairments, underscoring CRISPR-Cas9’s suitability for studying gene functions in erythropoiesis. Furthermore, BCL11A knockdown via sgRNAs results in elevated gamma-globin expression, offering a promising therapeutic avenue for beta-hemoglobinopathies. HPLC analysis reveals a substantial increase in HbF levels, particularly upon Znf4 deletion, emphasizing BCL11A gene potential as a therapeutic target. These findings also highlight the connection between the function of BCL11A and its structural organization, which can be modulated, and this insight can potentially be extended to uncover its roles in various other domains.