Actin Contributes to the Hyperexpression of Baculovirus Polyhedrin (polh) and p10 as a Component of Transcription Initiation Complex (TIC)

Hyperexpression of polh and p10, two very late genes, is one of the remarkable characteristics in the baculovirus life cycle. However, the mechanisms underlying the hyperexpression of these two genes are still incompletely understood. In this study, actin was identified as a highly potential binding partner of polh and p10 promoters by conducting DNA pull-down and LC–MS/MS analyses. Inhibiting actin dynamics delayed and decreased the transcription of polh and p10. Actin interacted with viral RNA polymerase and transcription regulators, and the nuclear import of viral polymerase was inhibited with the disruption of actin dynamics. Simultaneously, the high enrichment of actin in polh and p10 promoters discovered via a chromatin immunoprecipitation (ChIP) assay indicated that actin was a component of the viral polymerase TIC. Moreover, overexpression of actin surprisingly upregulated the expression of luciferase (Luc) under the control of polh and p10 promoters. Taken together, actin participated in the hyperexpression of polh and p10 as a component of TIC. These results facilitate the promotion of the expression efficiency of foreign genes in the baculovirus expression vector system (BEVS).

In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, natural antibodies isolated from convalescent patients are vulnerable to SARS-CoV-2 Spike mutations. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb dimer, named Nb1-Nb2, with tight affinity and super wide neutralization breadth against multiple SARS-CoV-2 variants of concern or interest. Deep-mutational scanning experiments identify the potential binding epitopes of the monomeric Nb1 and Nb2 on the RBD and demonstrate that bivalent Nb1-Nb2 has a strong escape resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that Nb1-Nb2 broadly neutralizes SARS-CoV-2, including variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1) and Mu (B.1.621). Furthermore, a heavy chain antibody is constructed by fusing the human IgG1 Fc to the biparatopic Nb (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0*10E-12 M) and neutralizing activity (IC50 = 0.0017 nM). Together, we developed a biparatopic human heavy chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

Bile Acids Gate Dopamine Transporter Mediated Currents

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Sterols such as cholesterol interact with monoamine transporters, including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward. This study explores the interactions of the BA, obeticholic acid (OCA), with DAT and characterizes the regulation of DAT activity via both electrophysiology and molecular modeling. We expressed murine DAT (mDAT) in Xenopus laevis oocytes and confirmed its functionality. Next, we showed that OCA promotes a DAT-mediated inward current that is Na+-dependent and not regulated by intracellular calcium. The current induced by OCA was transient in nature, returning to baseline in the continued presence of the BA. OCA also transiently blocked the DAT-mediated Li+-leak current, a feature that parallels DA action and indicates direct binding to the transporter in the absence of Na+. Interestingly, OCA did not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, regarding DA. Docking simulations performed for investigating the molecular mechanism of OCA action on DAT activity revealed two potential binding sites. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., J. Biol. Chem., 2014, 289, 25764–25773; Cheng and Bahar, Structure, 2015, 23, 2171–2181). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is coordinated by the DAT residues R445 and D436. Binding to that site may stabilize the inward-facing (IF) open state by preventing the re-formation of the IF-gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

The objects and effects of non-party intervention before the International Court of Justice

The International Court of Justice recognized the legitimacy of ‘non-party intervention’ under Article 62 of the Statute in its 1990 landmark decision on Nicaragua’s intervention in the Land, Island and Maritime Frontier Dispute (El Salvador v. Honduras). Such form of intervention ‘is not intended to enable a third State to tack on a new case, to become a new party, and so have its own claims adjudicated by the Court’. Its purpose is ‘protecting a State’s “interest of a legal nature” that might be affected by a decision in an existing case’. Whereas non-party intervention under Article 62 now forms part of the law in action within the Court’s system, its precise features and regime remain uncertain. Doubts concern the identification of its precise objects and the potential binding effects for a non-party intervener of the judgment issued between the original parties. The present article explores these issues in the light of the Court’s case law and state practice. It demonstrates that non-party intervention can have various potential objects, depending on how the intervener intends to influence the future judgment between the original parties. Building on the identification of these objects, it then questions the traditional construction denying any binding effect of the decision for a non-party intervener and argues that a judgment issued following intervention is binding as between the original parties and the intervener in so far as this judgment, whether expressly or by implication, decides issues related to the object of intervention.

CRISPR/Cas9 genome-wide screening identifies LUC7L2 that promotes radioresistance via autophagy in nasopharyngeal carcinoma cells

Radioresistance emerges as the major obstacle to nasopharyngeal carcinoma (NPC) treatment, further understanding of underlying mechanisms is necessary to overcome the radioresistance and improve the therapeutic effect. In this study, we first identified a candidate radioresistant-related gene LUC7L2 via CRISPR/Cas9 high-throughput screening and quantitative proteomic approach. Overexpression of LUC7L2 in NPC cells promoted cell viability following exposure to ionizing radiation (IR), while knockdown of LUC7L2 significantly slowed down the DNA replication and impaired cell survival, sensitized NPC-radioresistant cells to IR. Using immunoprecipitation assay, we found SQSTM1, an autophagy receptor, was a potential binding partner of LUC7L2. Down-regulation of LUC7L2 in NPC-radioresistant cells led to reduction of SQSTM1 expression and enhancement of autophagy level. Furthermore, LUC7L2 knockdown in combination with autophagy inhibitor, chloroquine (CQ), resulted in more NPC-radioresistant cell death. Besides, LUC7L2 was obviously distributed in NPC tissues, and high LUC7L2 expression correlated with shorter survival in NPC patients. Our data suggest that LUC7L2 plays a huge part in regulating radioresistance of NPC cells, and serves as a promising therapeutic target in re-sensitizing NPC to radiotherapy.

Utilization of Pectin from Okra as Binding Agent in Immediate Release Tablets

Polymeric materials from plants continue to be of interest to pharmaceutical scientists as potential binders in immediate release tablets due to availability, sustainability, and constant supply to feed local pharmaceutical industries. Paracetamol tablet formulations were utilized in investigating the potential binding characteristics of pectin harnessed from various okra genotypes (PC1-PC5) in Ghana. The pectin yields from the different genotypes ranged from 6.12 to 18.84%w/w. The pH of extracted pectin ranged from 6.39 to 6.92, and it had good swelling indices and a low moisture content. Pectin extracted from all genotypes were evaluated as binders (10, 15, and 20%w/v) and compared to tragacanth BP. All formulated tablets (F1-F18) passed the weight uniformity, drug content, hardness, and friability tests. Based on their crushing strength, tablets prepared with pectin from the various genotypes were relatively harder ( P ≤ 0.05 ) than tablets prepared with tragacanth BP. Tablets prepared with pectins as binders at 10%w/v and 15%w/v passed the disintegration and dissolution tests with the exception of PC4 at 15%w/v. Incorporation of pectin from all genotypes (excluding PC5) as a binder at concentrations above 15%w/v (F13, F16, F14, and F15) produced tablets which failed the disintegration test and showed poor dissolution profiles. Thus, pectin from these genotypes can be industrially commodified as binders in immediate release tablets using varying concentrations.

Evaluation of the binding and disintegrating properties of gum obtained from the stem bark of Cinnamomum zeylanicum

Excipients are the various ingredients, apart from the active pharmaceutical ingredients, which are added to pharmaceutical formulations. Excipients obtained from natural sources are preferred over those from synthetic sources because they are cheap, biocompatible and readily available. Gums are made up of carbohydrate units which are linked by glycosidic bonds. This study was aimed at evaluating the potential binding and disintegrating properties of gum obtained from the bark of Cinnamomum zeylanicum, which was obtained from Effiduase in the Ashanti region of Ghana. The gum was extracted using 96% ethanol and the moisture content, Fourier transform infrared spectroscopy spectra, water holding capacity, swelling index and flow properties of the gum were determined. The gum was used to formulate tablets at different concentrations (10% w/v, 15% w/v and 20% w/v) as binder with acacia as the standard. The gum was also used to formulate tablets at different concentrations (5% w/v, 7.5% w/v and 10% w/v) as disintegrant with starch as the standard. Quality control tests were then conducted on all formulated tablets. The gum exhibited good flow and physicochemical properties. All formulated tablets passed the uniformity of weight test, friability test, disintegration test, hardness test, uniformity of dimensions test and drug content. All batches of tablets, except Batch 7, passed the dissolution test. Based on the study carried out, C. zeylanicum gum can be used as an alternative excipient to acacia and starch as a binder and a disintegrant, respectively.

Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy

Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in the autophagy pathway, the so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B−/−) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.

Effect of Tetraphenylborate on Physicochemical Properties of Bovine Serum Albumin

The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]−) have been investigated by a set of experimental methods (isothermal titration calorimetry, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy) and molecular dynamics-based computational approaches. Two sets of structurally distinctive binding sites in BSA were found under the experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K). The obtained results, supported by the competitive interactions experiments of SDS with [B(Ph)4]− for BSA, enabled us to find the potential binding sites in BSA. The first site is located in the subdomain I A of the protein and binds two [B(Ph)4]− ions (logK(ITC)1 = 7.09 ± 0.10; ΔG(ITC)1 = −9.67 ± 0.14 kcal mol−1; ΔH(ITC)1 = −3.14 ± 0.12 kcal mol−1; TΔS(ITC)1 = −6.53 kcal mol−1), whereas the second site is localized in the subdomain III A and binds five ions (logK(ITC)2 = 5.39 ± 0.06; ΔG(ITC)2 = −7.35 ± 0.09 kcal mol−1; ΔH(ITC)2 = 4.00 ± 0.14 kcal mol−1; TΔS(ITC)2 = 11.3 kcal mol−1). The formation of the {[B(Ph)4]−}–BSA complex results in an increase in the thermal stability of the alfa-helical content, correlating with the saturation of the particular BSA binding sites, thus hindering its thermal unfolding.