Albumin-EDTA-Vanadium Is a Powerful Anti-Proliferative Agent, Following Entrance into Glioma Cells via Caveolae-Mediated Endocytosis

Human serum albumin (HSA) is efficiently taken up by cancer cells as a source of carbon and energy. In this study, we prepared a monomodified derivative of HSA covalently linked to an EDTA derivative and investigated its efficacy to shuttle weakly anti-proliferative EDTA associating ligands such as vanadium, into a cancer cell line. HSA-S-MAL-(CH2)2-NH-CO-EDTA was found to associate both with the vanadium anion (+5) and the vanadium cation (+4) with more than thrice the associating affinity of those ligands toward EDTA. Both conjugates internalized into glioma tumor cell line via caveolae-mediated endocytosis pathway and showed potent anti-proliferative capacities. IC50 values were in the range of 0.2 to 0.3 µM, potentiating the anti-proliferative efficacies of vanadium (+4) and vanadium (+5) twenty to thirty fold, respectively. HSA-EDTA-VO++ in particular is a cancer permeable prodrug conjugate. The associated vanadium (+4) is not released, nor is it active anti-proliferatively prior to its engagement with the cancerous cells. The bound vanadium (+4) dissociates from the conjugate under acidic conditions with half maximal value at pH 5.8. In conclusion, the anti-proliferative activity feature of vanadium can be amplified and directed toward a cancer cell line. This is accomplished using a specially designed HSA-EDTA-shuttling vehicle, enabling vanadium to be anti-proliferatively active at the low micromolar range of concentration.

Orthogonal genome-wide screens of bat cells identify MTHFD1 as a target of broad antiviral therapy

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.

Nanodissected elastically loaded clathrin lattices relax to increased curvature

Clathrin-mediated endocytosis (CME) is the major endocytosis pathway for the specific internalization of large compounds, growth factors, and receptors. Formation of internalized vesicles from the flat plasma membrane is accompanied by maturation of cytoplasmic clathrin coats. How clathrin coats mature and the mechanistic role of clathrin coats are still largely unknown. Maturation models proposed clathrin coats to mature at constant radius or constant area, driven by molecular actions or elastic energy. Here, combining high-speed atomic force microscopy (HS-AFM) imaging, HS-AFM nanodissection, and elasticity theory, we show that clathrin lattices deviating from the intrinsic curvature of clathrin form elastically loaded assemblies. Upon nanodissection of the clathrin network, the stored elastic energy in these lattices drives lattice relaxation to accommodate an ideal area-curvature ratio toward the formation of closed clathrin-coated vesicles. Our work supports that the release of elastic energy stored in curvature-frustrated clathrin lattices could play a major role in CME.

pH Sensitive Dextran Coated Fluorescent Nanodiamonds as a Biomarker for HeLa Cells Endocytic Pathway and Increased Cellular Uptake

Fluorescent nanodiamonds are a useful for biosensing of intracellular signaling networks or environmental changes (such as temperature, pH or free radical generation). HeLa cells are interesting to study with these nanodiamonds since they are a model cell system that is widely used to study cancer-related diseases. However, they only internalize low numbers of nanodiamond particles very slowly via the endocytosis pathway. In this work, we show that pH-sensitive, dextran-coated fluorescent nanodiamonds can be used to visualise this pathway. Additionally, this coating improved diamond uptake in HeLa cells by 5.3 times (*** p < 0.0001) and decreased the required time for uptake to only 30 min. We demonstrated further that nanodiamonds enter HeLa cells via endolysosomes and are eventually expelled by cells.

Hyaluronic Acid Derivative Effect on Niosomal Coating and Interaction with Cellular Mimetic Membranes

Hyaluronic acid (HA) is one of the most used biopolymers in the development of drug delivery systems, due to its biocompatibility, biodegradability, non-immunogenicity and intrinsic-targeting properties. HA specifically binds to CD44; this property combined to the EPR effect could provide an option for reinforced active tumor targeting by nanocarriers, improving drug uptake by the cancer cells via the HA-CD44 receptor-mediated endocytosis pathway. Moreover, HA can be easily chemically modified to tailor its physico-chemical properties in view of specific applications. The derivatization with cholesterol confers to HA an amphiphilic character, and then the ability of anchoring to niosomes. HA-Chol was then used to coat Span® or Tween® niosomes providing them with an intrinsic targeting shell. The nanocarrier physico-chemical properties were analyzed in terms of hydrodynamic diameter, ζ-potential, and bilayer structural features to evaluate the difference between naked and HA-coated niosomes. Niosomes stability was evaluated over time and in bovine serum. Moreover, interaction properties of HA-coated nanovesicles with model membranes, namely liposomes, were studied, to obtain insights on their interaction behavior with biological membranes in future experiments. The obtained coated systems showed good chemical physical features and represent a good opportunity to carry out active targeting strategies.

Overexpression of Toll-like receptor 4 contributes to the internalization and elimination of Escherichia coli in sheep by enhancing caveolae-dependent endocytosis

Background Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health. Toll-like receptor 4 (TLR4) plays a crucial role in host defence against Gram-negative bacteria. Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections. In a previous study, we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance. The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood. Results In the present study, we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model. The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection. This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis. Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4, Src signalling, or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria, weakened the ability of the monocytes to eliminate the bacteria, and increased the pH of the endosomes. Conclusion Together, our results reveal the effects of TLR4 on the control of E. coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model, providing theoretical support for breeding disease resistance in the future. Furthermore, Src and caveolin 1 (CAV1) could be potentially valuable targets for the control of infectious diseases.

Newcastle Disease Virus Entry into Chicken Macrophages via a pH-Dependent, Dynamin and Caveola-Mediated Endocytic Pathway That Requires Rab5

The cellular entry pathways and the mechanisms of Newcastle disease virus (NDV) entry into cells are poorly characterized. In this study, we demonstrated that chicken interferon-induced transmembrane protein 1 (chIFITM1) which is located in the early endosomes could limit the replication of NDV in chicken macrophage cell line HD11, suggesting the endocytic entry of NDV into chicken macrophages. Then, we presented a systematic study about the entry mechanism of NDV into chicken macrophages. First, we demonstrated that a low-pH condition and dynamin were required during NDV entry. However, NDV entry into chicken macrophages was independent of clathrin-mediated endocytosis. We also found that NDV entry was dependent on membrane cholesterol. The NDV entry and replication were significantly reduced by nystatin and Phorbol 12-myristate 13-acetate treatment, overexpression of dominant negative (DN) caveolin-1 or knockdown of caveolin-1, suggesting that NDV entry depends on caveola-mediated endocytosis. However, macropinocytosis did not play a role in NDV entry into chicken macrophages. Additionally, we found that Rab5, rather than Rab7, was involved in the entry and traffic of NDV. The colocalization of NDV with Rab5 and early endosome suggested that NDV virion was transported to early endosomes in a Rab5-dependent manner after internalization. Of particular note, the caveola-mediated endocytosis was also utilized by NDV to enter primary chicken macrophages. And NDV entered different cell types using different pathways. Collectively, our findings demonstrate for the first time that NDV virion enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway and Rab5 is involved in the traffic and location of NDV. IMPORTANCE Although the pathogenesis of Newcastle disease virus (NDV) has been extensively studied, the detailed mechanism of NDV entry into host cells is largely unknown. Macrophages are the first-line defenders of host defense against infection of pathogens. Chicken macrophages are considered as one of the main types of target cells during NDV infection. Here, we comprehensively investigated the entry mechanism of NDV in chicken macrophages. This is the first report to demonstrate that NDV enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway that requires Rab5. The result is important for our understanding of the entry of NDV in chicken macrophages, which will further advance the knowledge of NDV pathogenesis and provide useful clues for the development of novel preventive or therapeutic strategies against NDV infection. In addition, this information will contribute to our further understanding of pathogenesis with regard to other numbers of Avulavirus genus in Paramyxoviridae family.

Understanding In Vivo Fate of Nucleic Acid and Gene Medicines for the Rational Design of Drugs

Nucleic acid and genetic medicines are increasingly being developed, owing to their potential to treat a variety of intractable diseases. A comprehensive understanding of the in vivo fate of these agents is vital for the rational design, discovery, and fast and straightforward development of the drugs. In case of intravascular administration of nucleic acids and genetic medicines, interaction with blood components, especially plasma proteins, is unavoidable. However, on the flip side, such interaction can be utilized wisely to manipulate the pharmacokinetics of the agents. In other words, plasma protein binding can help in suppressing the elimination of nucleic acids from the blood stream and deliver naked oligonucleotides and gene carriers into target cells. To control the distribution of these agents in the body, the ligand conjugation method is widely applied. It is also important to understand intracellular localization. In this context, endocytosis pathway, endosomal escape, and nuclear transport should be considered and discussed. Encapsulated nucleic acids and genes must be dissociated from the carriers to exert their activity. In this review, we summarize the in vivo fate of nucleic acid and gene medicines and provide guidelines for the rational design of drugs.