Curcumin-Poly(sodium 4-styrenesulfonate) Conjugates as Potent Zika Virus Entry Inhibitors.

Curcumin, a natural product with recognized antiviral properties, is limited in its application largely due to its poor solubility. This study presents the synthesis of water-soluble curcumin-poly(sodium 4-styrenesulfonate) (Cur-PSSNan) covalent conjugates. The antiflaviviral activity of conjugates was validated in vitro by using the Zika virus as a model. In the development of these water-soluble curcumin-containing derivatives, we used the macromolecules reported by us to also hamper viral infections. Mechanistic investigations indicated that the conjugates exhibited excellent stability and bioavailability. The curcumin and macromolecules in concerted action interact directly with virus particles and block their attachment to host cells, hampering the infection process.

Cellular and population strategies underpinning neurotoxin production and sporulation in Clostridium botulinum type E cultures.

IMPORTANCE: Toxin production and sporulation are key determinants of pathogenesis in Clostridia. Toxins cause the clinical manifestation of clostridial diseases, including diarrhea and colitis, tissue damage, and systemic effects on the nervous system. Spores ensure long-term survival and persistence in the environment, act as infectious agents, and initiate the host tissue colonization leading to infection. Understanding the interplay between toxin production and sporulation and their coordination in bacterial cells and cultures provides novel intervention points for controlling the public health and food safety risks caused by clostridial diseases. We demonstrate environmentally driven cellular heterogeneity in botulinum neurotoxin and spore production in Clostridium botulinum type E populations and discuss the biological rationale of toxin and spore production in the pathogenicity and ecology of C. botulinum. The results invite to reassess the epidemiology of botulism and may have important implications in the risk assessment and risk management strategies in food processing and human and animal health.

Reversible rearrangement of the cellular cytoskeleton: A key to the broad-spectrum antiviral activity of novel amphiphilic polymers.

The battle against emerging viral infections has been uneven, as there is currently no broad-spectrum drug available to contain the spread of novel pathogens throughout the population. Consequently, the pandemic outbreak that occurred in early 2020 laid bare the almost empty state of the pandemic box. Therefore, the development of novel treatments with broad specificity has become a paramount concern in this post-pandemic era. Here, we propose copolymers of poly (sodium 2-(acrylamido)-2-methyl-1-propanesulfonate) (PAMPS) and poly (sodium 11-(acrylamido)undecanoate (AaU), both block (PAMPS75-b-PAaUn) and random (P(AMPSm-co-AaUn)) that show efficacy against a broad range of alpha and betacoronaviruses. Owing to their intricate architecture, these polymers exhibit a highly distinctive mode of action, modulating nano-mechanical properties of cells and thereby influencing viral replication. Through the employment of confocal and atomic force microscopy techniques, we discerned perturbations in actin and vimentin filaments, which correlated with modification of cellular elasticity and reduction of glycocalyx layer. Intriguingly, this process was reversible upon polymer removal from the cells. To ascertain the applicability of our findings, we assessed the efficacy and underlying mechanism of the inhibitors using fully differentiated human airway epithelial cultures, wherein near-complete abrogation of viral replication was documented. Given their mode of action, these polymers can be classified as biologically active nanomaterials that exploit a highly conserved molecular target-cellular plasticity-proffering the potential for truly broad-spectrum activity while concurrently for drug resistance development is minimal.

Novel inhibitors of HSV-1 protease effective in vitro and in vivo.

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen known to cause infections of diverse severity, ranging from mild ulceration of mucosal and dermal tissues to life-threatening viral encephalitis. In most cases, standard treatment with acyclovir is sufficient to manage the disease progression. However, the emergence of ACV-resistant strains drives the need for new therapeutics and molecular targets. HSV-1 VP24 is a protease indispensable for the assembly of mature virions and, as such, constitutes an interesting target for the therapy. In this study, we present novel compounds, KI207M and EWDI/39/55BF, that block the activity of VP24 protease and consequently inhibit HSV-1 infection in vitro and in vivo. The inhibitors were shown to prevent the egress of viral capsids from the cell nucleus and suppress the cell-to-cell spread of the infection. They were also proven effective against ACV-resistant HSV-1 strains. Considering their low toxicity and high antiviral potency, the novel VP24 inhibitors could provide an alternative for treating ACV-resistant infections or a drug to be used in combined, highly effective therapy.

Poly(ethylene glycol)-block-poly(sodium 4-styrenesulfonate) Copolymers as Efficient Zika Virus Inhibitors: In Vitro Studies.

A series of poly(ethylene glycol)-block-poly(sodium 4-styrenesulfonate) (PEG-b-PSSNa) copolymers were synthesized, and their antiviral activity against Zika virus (ZIKV) was determined. The polymers inhibit ZIKV replication in vitro in mammalian cells at nontoxic concentrations. The mechanistic analysis revealed that the PEG-b-PSSNa copolymers interact directly with viral particles in a zipper-like mechanism, hindering their interaction with the permissive cell. The antiviral activity of the copolymers is well-correlated with the length of the PSSNa block, indicating that the copolymers' ionic blocks are biologically active. The blocks of PEG present in copolymers studied do not hinder that interaction. Considering the practical application of PEG-b-PSSNa and the electrostatic nature of the inhibition, the interaction between the copolymers and human serum albumin (HSA) was evaluated. The formation of PEG-b-PSSNa-HSA complexes in the form of negatively charged nanoparticles well-dispersed in buffer solution was observed. That observation is promising, given the possible practical application of the copolymers.

Crystal structures of OrfX1, OrfX2 and the OrfX1-OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1.

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A-G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1-OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.

Self-Organized Nanoparticles of Random and Block Copolymers of Sodium 2-(Acrylamido)-2-methyl-1-propanesulfonate and Sodium 11-(Acrylamido)undecanoate as Safe and Effective Zika Virus Inhibitors.

A series of anionic homopolymers, poly(sodium 2-(acrylamido)-2-methyl-1-propanesulfonate) (PAMPS) and amphiphilic copolymers of AMPS and sodium 11-(acrylamido)undecanoate (AaU), both block (PAMPS75-b-PAaUn), and random (P(AMPSm-co-AaUn)), were synthesized and their antiviral activity against Zika virus (ZIKV) was evaluated. Interestingly, while the homopolymers showed limited antiviral activity, the copolymers are very efficient antivirals. This observation was explained considering that under the conditions relevant to the biological experiments (pH 7.4 PBS buffer) the macromolecules of these copolymers exist as negatively charged (zeta potential about -25 mV) nanoparticles (4-12 nm) due to their self-organization. They inhibit the ZIKV replication cycle by binding to the cell surface and thereby blocking virus attachment to host cells. Considering good solubility in aqueous media, low toxicity, and high selectivity index (SI) of the PAMPS-b-PAaU copolymers, they can be considered promising agents against ZIKV infections.

Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid.

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.

Magnetic Properties of Collagen-Chitosan Hybrid Materials with Immobilized Superparamagnetic Iron Oxide Nanoparticles (SPIONs).

The paper presents results of our studies on hybrid materials based on polymers of natural origin containing superparamagnetic iron oxide nanoparticles (SPIONs). Such nanoparticles, coated with the chitosan derivative, were immobilized in a chitosan-collagen hydrogel matrix by crosslinking with genipin. Three types of biopolymer matrices of different collagen-to-chitosan ratios were studied. A thorough magnetic characterization was performed, including magnetic susceptibility, magnetization, and hysteresis loop measurements in a temperature range of 4 K to 300 K and a magnetic field induction up to 8 Tesla. The effect of SPION immobilization and material composition on the magnetic properties of the hybrids was investigated. The results showed that hybrid materials with covalently bounded SPIONs preserved the superparamagnetic character of SPIONs and exhibited promising magnetic properties, which are important for their potential applications.

Addressing the Osteoporosis Problem-Multifunctional Injectable Hybrid Materials for Controlling Local Bone Tissue Remodeling.

Novel multifunctional biomimetic injectable hybrid systems were synthesized. The physicochemical as well as biological in vitro and in vivo tests demonstrated that they are promising candidates for bone tissue regeneration. The hybrids are composed of a biopolymeric collagen/chitosan/hyaluronic acid matrix and amine group-functionalized silica particles decorated with apatite to which the alendronate molecules were coordinated. The components of these systems were integrated and stabilized by cross-linking with genipin, a compound of natural origin. They can be precisely injected into the diseased tissue in the form of a viscous sol or a partially cross-linked hydrogel, where they can serve as scaffolds for locally controlled bone tissue regeneration/remodeling by supporting the osteoblast formation/proliferation and maintaining the optimal osteoclast level. These materials lack systemic toxicity. They can be particularly useful for the repair of small osteoporotic bone defects.

Tuning the Surface Properties of Poly(Allylamine Hydrochloride)-Based Multilayer Films.

The layer-by-layer (LbL) method of polyelectrolyte multilayer (PEM) fabrication is extremely versatile. It allows using a pair of any oppositely charged polyelectrolytes. Nevertheless, it may be difficult to ascribe a particular physicochemical property of the resulting PEM to a structural or chemical feature of a single component. A solution to this problem is based on the application of a polycation and a polyanion obtained by proper modification of the same parent polymer. Polyelectrolyte multilayers (PEMs) were prepared using the LbL technique from hydrophilic and amphiphilic derivatives of poly(allylamine hydrochloride) (PAH). PAH derivatives were obtained by the substitution of amine groups in PAH with sulfonate, ammonium, and hydrophobic groups. The PEMs were stable in 1 M NaCl and showed three different modes of thickness growth: exponential, mixed exponential-linear, and linear. Their surfaces ranged from very hydrophilic to hydrophobic. Root mean square (RMS) roughness was very variable and depended on the PEM composition, sample environment (dry, wet), and the polymer constituting the topmost layer. Atomic force microscopy (AFM) imaging of the surfaces showed very different morphologies of PEMs, including very smooth, porous, and structured PEMs with micellar aggregates. Thus, by proper choice of PAH derivatives, surfaces with different physicochemical features (growth type, thickness, charge, wettability, roughness, surface morphology) were obtained.

CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research.

The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt "bookmark" sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and "bookmark"-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique "watermark" through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.

HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV.

Among seven coronaviruses that infect humans, three (severe acute respiratory syndrome coronavirus [SARS-CoV], Middle East respiratory syndrome coronavirus [MERS-CoV], and the newly identified severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) are associated with a severe, life-threatening respiratory infection and multiorgan failure. We previously proposed that the cationically modified chitosan N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) is a potent inhibitor of human coronavirus NL63 (HCoV-NL63). Next, we demonstrated the broad-spectrum antiviral activity of the compound, as it inhibited all low-pathogenicity human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1). Here, using in vitro and ex vivo models of human airway epithelia, we show that HTCC effectively blocks MERS-CoV and SARS-CoV-2 infection. We also confirmed the mechanism of action for these two viruses, showing that the polymer blocks the virus entry into the host cell by interaction with the S protein.IMPORTANCE The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the health care measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of an HTCC compound, previously developed by us, which may be used as a potential inhibitor of currently circulating highly pathogenic coronaviruses-SARS-CoV-2 and MERS-CoV.

In Vitro Inhibition of Zika Virus Replication with Poly(Sodium 4-Styrenesulfonate).

Zika virus (ZIKV) is an emerging mosquito-borne pathogen associated with microcephaly and other congenital abnormalities in newborns as well as neurologic complications in adults. The explosive transmission of the virus in the last ten years put it in the limelight and improved our understanding of its biology and pathology. Currently, no vaccine or drugs are available to prevent or treat ZIKV infections. Knowing the potential of flaviviruses to broaden their geographic distribution, as observed for the West Nile virus, it is of importance to develop novel antiviral strategies. In this work, we identified poly(sodium 4-styrenesulfonate) (PSSNa) as a new polymeric ZIKV inhibitor. We demonstrated that PSSNa inhibits ZIKV replication in vitro both in animal and human cells, while no cytotoxicity is observed. Our mechanistic studies indicated that PSSNa acts mostly through direct binding to ZIKV particle and blocking its attachment to the host cells.

Stabilization of liposomes with silicone layer improves their elastomechanical properties while not compromising biological features.

The liposomes are among the most promising types of drug delivery systems but low stability significantly limits their application. Some approaches proposed to overcome this drawback may affect the liposomes toxicity profile. It is assumed that developed by us and presented here stabilization method involving formation of silicone network within the liposomal bilayer will improve elastomechanical properties of vesicles while not deteriorating their biocompatibility. The silicone-stabilized liposomes were prepared by base-catalyzed polycondensation process of the 1,3,5,7-tetramethylcyclotetrasiloxane (D4H) within the liposomal bilayer. The systematic biological in vitro studies of vesicles obtained were carried out. Moreover, the elastomechanical features investigation employing atomic force microscopy (AFM) measurements was performed. These properties of the liposome membrane are of great importance since they define the nanocarriers' stability as well as play a significant role in their cellular uptake via endocytosis. Applying the Derjaguin-Muller-Toporov (DMT) model, the elastic modulus of the silicone-stabilized liposomes was determined and compared to that characteristic for the pristine liposomes. The in vitro biological evaluation of silicone-stabilized liposomes demonstrated that these vesicles are not toxic for blood cells isolated from healthy donors and they do not induce oxidative stress in HepG2 cells. AFM results confirmed the stabilizing effect of silicone and revealed that the silicone network improves the elastomechanical properties of the resulted liposomes. This is the first report demonstrating that the silicone-stabilized liposomes retain biocompatibility of pristine liposomes' while acquire significantly better elastomechanical features.

Pioglitazone-Loaded Nanostructured Hybrid Material for Skin Ulcer Treatment.

Pioglitazone, a popular antidiabetic drug, which was recently shown to be effective in the treatment of skin ulcers, was successfully encapsulated in polysaccharide nanoparticles and used as a bioactive component of the wound-dressing material based on modified bacterial nanocellulose. Alginate and hydroxypropyl cellulose were used as a matrix for the nanoparticulate drug-delivery system. The matrix composition and particles' size, as well as drug encapsulation efficiency and loading, were optimized. Pioglitazone hydrochloride (PIO) loaded particles were coated with chitosan introduced into the crosslinking medium, and covalently attached to the surface of bacterial nanocellulose functionalized with carboxyl groups. PIO was released from the surface of the hybrid material in a controlled manner for 5 days. Preliminary cytotoxicity studies confirmed safety of the system at PIO concentrations as high as 20 mg/mL. The obtained hybrid system may have potential application in the treatment of skin ulcers e.g., in diabetic foot.

Berberine Hampers Influenza A Replication through Inhibition of MAPK/ERK Pathway.

BACKGROUND: Berberine (BBR) is an isoquinoline alkaloid which exhibits a variety of biological and therapeutic properties, and has been reported by some to block replication of the influenza virus. However, contradictory results have also been presented, and the mechanistic explanation is lacking. METHODS: A panel of cell lines (Madin-Darby canine kidney (MDCK), adenocarcinoma human alveolar basal epithelial cells (A549), lung epithelial type I (LET1)) and primary human airway epithelial cells (HAE) susceptible to influenza virus infection were infected with a seasonal influenza A virus in the presence or absence of BBR. Cytotoxicity towards cell lines was measured using XTT assay. The yield of the virus was analyzed using RT-qPCR. To study the molecular mechanism of BBR, confocal microscopy and Western blot analyses of cellular fractions were applied. RESULTS AND CONCLUSIONS: Our results show cell-type-dependent anti-influenza properties of BBR in vitro which suggests that the compound acts on the cell and not the virus. Importantly, BBR hampers influenza replication in primary human airway epithelium 3D cultures that mimic the natural replication site of the virus. Studies show that the influenza A virus upregulates the mitogen-activated protein kinase/extracellular signal-related kinase (MAPK/ERK) pathway and hijacks this pathway for nucleolar export of the viral ribonucleoprotein. Our results suggest that BBR interferes with this process and hampers influenza A replication.

Near-Infrared Photoactive Aza-BODIPY: Thermally Robust and Photostable Photosensitizer and Efficient Electron Donor.

We report herein the synthesis of aza-BODIPY substituted with strongly electron-donating p-(diphenylamino)phenyl substituents (p-Ph2 N-) at 3,5-positions. The presence of p-Ph2 N- groups lowers the energy of the singlet excited state (Es ) to 1.48 eV and induces NIR absorption with λabs at 789 nm in THF. The compound studied is weakly emissive with the emission band (λf ) at 837 nm and with the singlet lifetime (τS ) equal to 100 ps. Nanosecond laser photolysis experiments of the aza-BODIPY in question revealed T1 →Tn absorption spanning from ca. 350-550 nm with the triplet lifetime (τT ) equal to 21 µs. By introducing a heavy atom (Br) into the structure of the aza-BODIPY, we managed to turn it into a NIR operating photosensitizer. The photosensitized oxygenation of the model compound-diphenylisobenzofuran (DPBF)-proceedes via Type I and/or Type III mechanism without formation of singlet oxygen (1 O2 ). As estimated by CV/DPV measurements, the p-Ph2 N- substituted aza-BODIPYs studied exhibits oxidation processes at relatively low oxidation potentials (Eox1 ), pointing to the very good electron-donating properties of these molecules. Extremely high photostability and thermal robustness up to approximately 300 °C are observed for the p-Ph2 N- substituted aza-BODIPYs.

Improved Pharmacokinetics and Tissue Uptake of Complexed Daidzein in Rats.

The pharmacokinetic profile and tissue uptake of daidzein (DAI) was determined in rat serum and tissues (lungs, eyes, brain, heart, spleen, fat, liver, kidney, and testes) after intravenous and intraperitoneal administration of DAI in suspension or complexed with ethylenediamine-modified γ-cyclodextrin (GCD-EDA/DAI). The absolute and relative bioavailability of DAI suspended (20 mg/kg i.v. vs. 50 mg/kg i.p.) and complexed (0.54 mg/kg i.v. vs. 1.35 mg/kg i.p.) was determined. After i.p. administration, absorption of DAI complexed with GCD-EDA was more rapid (tmax = 15 min) than that of DAI in suspension (tmax = 45 min) with a ca. 3.6 times higher maximum concentration (Cmax = 615 vs. 173 ng/mL). The i.v. half-life of DAI was longer in GCD-EDA/DAI complex compared with DAI in suspension (t0.5 = 380 min vs. 230 min). The volume of distribution of DAI given i.v. in GCD-EDA/DAI complex was ca. 6 times larger than DAI in suspension (38.6 L/kg vs. 6.2 L/kg). Our data support the concept that the pharmacokinetics of DAI suspended in high doses are nonlinear. Increasing the intravenous dose 34 times resulted in a 5-fold increase in AUC. In turn, increasing the intraperitoneal dose 37 times resulted in a ca. 2-fold increase in AUC. The results of this study suggested that GCD-EDA complex may improve DAI bioavailability after i.p. administration. The absolute bioavailability of DAI in GCD-EDA inclusion complex was ca. 3 times greater (F = 82.4% vs. 28.2%), and the relative bioavailability was ca. 21 times higher than that of DAI in suspension, indicating the need to study DAI bioavailability after administration by routes other than intraperitoneal, e.g., orally, subcutaneously, or intramuscularly. The concentration of DAI released from GCD-EDA/DAI inclusion complex to all the rat tissues studied was higher than after administration of DAI in suspension. The concentration of DAI in brain and lungs was found to be almost 90 and 45 times higher, respectively, when administered in complex compared to the suspended DAI. Given the nonlinear relationship between DAI bioavailability and the dose released from the GCD-EDA complex, complexation of DAI may thus offer an effective approach to improve DAI delivery for treatment purposes, for example in mucopolysaccharidosis (MPS), allowing the reduction of ingested DAI doses.