Updating Zika Diagnostic Methods: The Point-of-Care Approach.

Zika virus (ZIKV) has gained great importance worldwide since the past epidemic that occurred in 2015 in Brazil. Early identification of ZIKV is critical to minimize transmission and prevents potentially devastating consequences, including microcephaly in neonates of infected women, congenital blindness, or Guillain-Barré Syndrome. However, this is not an easy task, considering that approximately 80% of ZIKV infection cases are asymptomatic or oligosymptomatic, there are diverse modes of transmission (vertical transmission is through vectors and horizontal transmission through blood, saliva, semen, and urine from infected people), and the fact that ZIKV has a high identity percentage with other cocirculating Flaviviruses such as dengue. Here, we review ZIKV diagnostic methods, with special emphasis on the development of point-of-care diagnostic assays, since these devices commonly have two important advantages: they provide prompt screening and are affordable.

Intraocular Penetration of a vNAR: In Vivo and In Vitro VEGF165 Neutralization.

Variable new antigen receptor domain (vNAR) antibodies are novel, naturally occurring antibodies that can be isolated from naïve, immune or synthetic shark libraries. These molecules are very interesting to the biotechnology and pharmaceutical industries because of their unique characteristics related to size and tissue penetrability. There have been some approved anti-angiogenic therapies for ophthalmic conditions, not related to vNAR. This includes biologics and chimeric proteins that neutralize vascular endothelial growth factor (VEGF)165, which are injected intravitreal, causing discomfort and increasing the possibility of infection. In this paper, we present a vNAR antibody against human recombinant VEGF165 (rhVEGF165) that was isolated from an immunized Heterodontus francisci shark. A vNAR called V13, neutralizes VEGF165 cytokine starting at 75 µg/mL in an in vitro assay based on co-culture of normal human dermal fibroblasts (NHDFs) and green fluorescence protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) cells. In the oxygen-induced retinopathy model in C57BL/6:Hsd mice, we demonstrate an endothelial cell count decrease. Further, we demonstrate the intraocular penetration after topical administration of 0.1 µg/mL of vNAR V13 by its detection in aqueous humor in New Zealand rabbits with healthy eyes after 3 h of application. These findings demonstrate the potential of topical application of vNAR V13 as a possible new drug candidate for vascular eye diseases.

vNARs as Neutralizing Intracellular Therapeutic Agents: Glioblastoma as a Target.

Glioblastoma is the most prevalent and fatal form of primary brain tumors. New targeted therapeutic strategies for this type of tumor are imperative given the dire prognosis for glioblastoma patients and the poor results of current multimodal therapy. Previously reported drawbacks of antibody-based therapeutics include the inability to translocate across the blood-brain barrier and reach intracellular targets due to their molecular weight. These disadvantages translate into poor target neutralization and cancer maintenance. Unlike conventional antibodies, vNARs can permeate tissues and recognize conformational or cryptic epitopes due to their stability, CDR3 amino acid sequence, and smaller molecular weight. Thus, vNARs represent a potential antibody format to use as intrabodies or soluble immunocarriers. This review comprehensively summarizes key intracellular pathways in glioblastoma cells that induce proliferation, progression, and cancer survival to determine a new potential targeted glioblastoma therapy based on previously reported vNARs. The results seek to support the next application of vNARs as single-domain antibody drug-conjugated therapies, which could overcome the disadvantages of conventional monoclonal antibodies and provide an innovative approach for glioblastoma treatment.

First pan-specific vNAR against human TGF-ß as a potential therapeutic application: in silico modeling assessment.

Immunotherapies based on antibody fragments have been developed and applied to human diseases, describing novel antibody formats. The vNAR domains have a potential therapeutic use related to their unique properties. This work used a non-immunized Heterodontus francisci shark library to obtain a vNAR with recognition of TGF-ß isoforms. The isolated vNAR T1 selected by phage display demonstrated binding of the vNAR T1 to TGF-ß isoforms (-ß1, -ß2, -ß3) by direct ELISA assay. These results are supported by using for the first time the Single-Cycle kinetics (SCK) method for Surface plasmon resonance (SPR) analysis for a vNAR. Also, the vNAR T1 shows an equilibrium dissociation constant (KD) of 9.61 × 10-8 M against rhTGF-ß1. Furthermore, the molecular docking analysis revealed that the vNAR T1 interacts with amino acid residues of TGF-ß1, which are essential for interaction with type I and II TGF-ß receptors. The vNAR T1 is the first pan-specific shark domain reported against the three hTGF-ß isoforms and a potential alternative to overcome the challenges related to the modulation of TGF-ß levels implicated in several human diseases such as fibrosis, cancer, and COVID-19.

Embedded Living HER2+ Cells in a 3D Gelatin-Alginate Hydrogel as an In Vitro Model for Immunotherapy Delivery for Breast Cancer.

Epidermal growth factor receptor 2 (HER2) is the second target molecule most commonly used in breast cancer treatment. Both recurrence and metastasis are still deadly for HER2+ breast cancer patients. Hydrogels can be an option for developing three-dimensional (3D) cell culture systems that resemble tumor features better than monolayer cultures and could be used for preclinical screening for new biotherapeutics. Biopolymers (gelatin and alginate) were used to develop a hydrogel capable of encapsulating living HER2+ breast cancer cells BT-474/GFP. The hydrogel was physicochemically characterized, and the viability of embedded cells was evaluated. The hydrogel developed had suitable physical properties, with swelling of 38% of its original mass at 20 h capacity and pore sizes between 20 and 125 µm that allowed cells to maintain their morphology in a 3D environment, in addition to being biocompatible and preserving 90% of cell viability at 10 days. Furthermore, encapsulated BT-474/GFP cells maintained HER2 expression that could be detected by the Trastuzumab-fluorescent antibody, so this hydrogel could be used to evaluate new HER2-targeted therapies.

In Vitro Antiviral Activity of Nordihydroguaiaretic Acid against SARS-CoV-2.

The coronavirus infectious disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has been spreading rapidly worldwide, creating a pandemic. This article describes the evaluation of the antiviral activity of nordihydroguaiaretic acid (NDGA), a molecule found in Creosote bush (Larrea tridentata) leaves, against SARS-CoV-2 in vitro. A 35 µM concentration of NDGA was not toxic to Vero cells and exhibited a remarkable inhibitory effect on the SARS-CoV-2 cytopathic effect, viral plaque formation, RNA replication, and expression of the SARS-CoV-2 spike glycoprotein. The 50% effective concentration for NDGA was as low as 16.97 µM. Our results show that NDGA could be a promising therapeutic candidate against SARS-CoV-2.

Possible Routes for Zika Virus Vertical Transmission in Human Placenta: A Comprehensive Review.

Zika virus (ZIKV) infections have gained notoriety due to congenital abnormalities. Pregnant women have a greater risk of ZIKV infection and consequent transmission to their progeny due to the immunological changes associated with pregnancy. ZIKV has been detected in amniotic fluid, as well as in fetal and neonatal tissues of infected pregnant women. However, the mechanism by which ZIKV reaches the fetus is not well understood. The four dengue virus serotypes have been the most widely used flaviviruses to elucidate the host-cell entry pathways. Nevertheless, it is of increasing interest to understand the specific interaction between ZIKV and the host cell, especially in the gestation period. Herein, the authors describe the mechanisms of prenatal vertical infection of ZIKV based on results from in vitro, in vivo, and ex vivo studies, including murine models and nonhuman primates. It also includes up-to-date knowledge from ex vivo and natural infections in pregnant women explaining the vertical transmission along four tracks: transplacental, paracellular, transcytosis mediated by extracellular vesicles, and paraplacental route and the antibody-dependent enhancement process. A global understanding of the diverse pathways used by ZIKV to cross the placental barrier and access the fetus, along with a better comprehension of the pathogenesis of ZIKV in pregnant females, may constitute a fundamental role in the design of antiviral drugs to reduce congenital disabilities associated with ZIKV.

3D Cell Culture Models in COVID-19 Times: A Review of 3D Technologies to Understand and Accelerate Therapeutic Drug Discovery.

In the last decades, emerging viruses have become a worldwide concern. The fast and extensive spread of the disease caused by SARS-CoV-2 (COVID-19) has impacted the economy and human activity worldwide, highlighting the human vulnerability to infectious diseases and the need to develop and optimize technologies to tackle them. The three-dimensional (3D) cell culture models emulate major tissue characteristics such as the in vivo virus-host interactions. These systems may help to generate a quick response to confront new viruses, establish a reliable evaluation of the pathophysiology, and contribute to therapeutic drug evaluation in pandemic situations such as the one that humanity is living through today. This review describes different types of 3D cell culture models, such as spheroids, scaffolds, organoids, and organs-on-a-chip, that are used in virus research, including those used to understand the new severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2).

Improvement of the wound healing properties of hydrogels with N-acetylcysteine through their modification with methacrylate-containing polymers.

Hydrogels with antioxidant activity have shown to significantly improve the standard of care, because they promote efficient wound healing, i.e. regeneration. N-Acetylcysteine (NAC) is an antioxidant amino acid derivative that promotes complete tissue restoration. However, NAC has anticoagulant properties that may also hinder blood coagulation, which is crucial for hydrogels for wound healing applications. To take advantage of the regenerative activity of NAC while avoiding hampering the hemostasis stage during wound healing, we modified gelatin-NAC with the methacrylate-containing polymers 2-hydroxyethyl methacrylate (H) and poly(ethylene glycol) methyl ether methacrylate (P) to produce Gel-HP-NAC. These hydrogels clotted more blood and faster than Gel and Gel-NAC hydrogels, while maintaining fluid absorption properties adequate to promote wound healing. Similarly, there were more viable human skin fibroblasts after 10 days cultured in Gel-HP-NAC compared with Gel and Gel-NAC. A mouse full-thickness skin wound model demonstrated that Gel-HP-NAC hydrogels improved the wound healing process as compared to the untreated group as proved by the increased wound closure rates and re-epithelialization. Histology of the biopsied tissues indicated more organized collagen deposits on the wounds treated with either Gel-HP-NAC or Gel-NAC than untreated wounds. Our results show that modification of NAC-containing hydrogels through methacrylate-containing polymers improved their wound healing properties, including blood-clotting, and demonstrate the potential of Gel-HP-NAC hydrogels for wound treatment and tissue regeneration.

Potential Therapeutic Applications of Synthetic Conotoxin s-cal14.2b, Derived from Californiconus californicus, for Treating Type 2 Diabetes.

The FDA's approval of peptide drugs such as Ziconotide or Exendin for pain relief and diabetes treatment, respectively, enhanced the interest to explore novel conotoxins from Conus species venom. In general, conotoxins can be used in pathologies where voltage-gated channels, membrane receptors, or ligands alter normal physiological functions, as in metabolic diseases such as Type 2 diabetes. In this study, the synthetic cal14.2b (s-cal14.2b) from the unusual Californiconus californicus demonstrated bioactivity on NIT-1 insulinoma cell lines stimulating insulin secretion detecting by high performance liquid chromatography (HPLC). Accordingly, s-cal14.2b increased the CaV1.2/1.3 channel-current by 35 ± 4% with a recovery τ of 10.3 ± 4 s in primary cell culture of rat pancreatic ß-cells. The in vivo results indicated a similar effect of insulin secretion on mice in the glucose tolerance curve model by reducing the glucose from 500 mg/dL to 106 mg/dL in 60 min, compared to the negative control of 325 mg/dL at the same time. The PET-SCAN with radiolabeling 99mTc-s-cal14.2b demonstrated biodistribution and accumulation in rat pancreas with complete depuration in 24 h. These findings show the potential therapeutic use of s-cal14.2b in endocrinal pathologies such as early stages of Type 2 Diabetes where the pancreas's capability to produce insulin is still effective.

Anti-fouling SERS-based immunosensor for point-of-care detection of the B7-H6 tumor biomarker in cervical cancer patient serum.

Herein, we report the development of sandwich type Surface Enhanced Raman Spectroscopy (SERS) immunosensor modified to be zwitterionic for the detection of soluble B7-H6 biomarker in blood serum from cervical cancer patients. Anti-fouling capture SERS substrate of biosensor based on gold (Au) thin film was modified with a self-assembled monolayer of zwitterionic l-cysteine to combat serum fouling and was then conjugated with NKp30 receptor protein to capture the B7-H6 biomarker in blood serum. The SERS nanoprobe based on spiky gold nanoparticles (AuNPs) was functionalized with ATP reporter molecule, that is stable at a wide range of pH, making the SERS signal reliable in complex media. Then, it was conjugated with anti-B7-H6 antibody forming the complex anti-B7-H6@ATP@AuNPs (i.e., SERS nanoprobe). The proposed immunosensor demonstrated high reproducibility for the quantitative detection of soluble tumor biomarker B7-H6 within the range of 10-10 M to 10-14 M with limit of detection (LOD) of 10-14 M or 10.8 fg mL-1, in the cancer patient serum, greatly exceeding (100 fold) the LOD of commercially available ELISA kits. Such low LOD is partially the result of zwitterionic modification which reduces the serum fouling by 55% compared to traditionally used BSA blocked capture substrates (i.e., control). Notably, this immunosensors demonstrated higher accuracy for detecting the B7-H6 biomarker in undiluted blood serum samples from cervical cancer patients and outperforms the currently available analytical techniques, making it reliable for point of care (POC) testing.

Novel anti-HER2 peptide-conjugated theranostic nanoliposomes combining NaYF4:Yb,Er nanoparticles for NIR-activated bioimaging and chemo-photodynamic therapy against breast cancer.

Herein, we reported the fabrication of novel peptide-conjugated ligand-targeted nanoliposomes (LTLs) for chemo-photodynamic therapy against HER2-positive breast cancer. The LTL core was utilized for encapsulating doxorubicin (DOX) for chemotherapy, and methylene blue (MB) attached NaYF4:Yb,Er upconversion nanoparticles (UCNPs) for NIR-activated bioimaging and leveraging its visible emission for photoexciting MB for enhanced photodynamic therapy (PDT). The specificity of our LTLs was achieved by conjugating a newly discovered anti-HER2 peptide screened from a phage display peptide library. The high selectivity of the peptide-conjugated LTLs was confirmed by confocal imaging of SKBR-3 (HER2-positive) and MCF-7 (HER2-negative) breast cancer cell lines, illustrating its target-specific nature. The energy transfer from UCNPs to MB was verified, thus enabling the generation of reactive oxygen species upon activation with a 975 nm laser source (0.60 W cm-2) under 5 min continuous excitation. A significant decline in the cell viability by 95% was observed using chemo-photodynamic combinational therapy, whereas for chemo-drug alone and PDT alone, the cell proliferation declined by 77% and 84%, respectively. Furthermore, we demonstrated an improved uptake of the LTLs inside a 3D model of SKBR-3 tumor spheroids, where the spheroid cell viability was suppressed by 66% after the use of combinational therapy. Thus, our results suggest great prospective use of theranostic LTLs for breast cancer management.

Effect of starch on the mechanical and in vitro properties of collagen-hydroxyapatite sponges for applications in dentistry.

This study sought to improve the mechanical and blood-absorbing properties of collagen sponges, while keeping them compressible, by incorporating blended hydroxyapatite (HA)-starch. Results were compared with CollaPlug(®) (pure collagen). The elastic modulus increased from 1.5±0.2kPa for CollaPlug(®) to 49±8kPa for sponges with composition 1:4:10 (collagen:HA:starch, by weight). The modified microstructure and surface area provided by the starch granules on the sponges improved cell viability. Sponges with composition 1:4:10 maintained their blood-clotting capability with almost no change from 5 to 15min after contact with blood, while CollaPlug(®) diminished to about half its capacity to absorb blood and form clots. Incorporation of HA-starch into the sponges with composition of 1:4:10, increased the elastic modulus of the collagen-HA sponges, making them more structurally robust. The viability of cells and the blood-clotting capability increased with starch incorporation.

Updating zika diagnostic methods: the point-of-care approach

Abstract Zika virus (ZIKV) has gained great importance worldwide since the past epidemic that occurred in 2015 in Brazil. Early identification of ZIKV is critical to minimize transmission and prevents potentially devastating consequences, including microcephaly in neonates of infected women, congenital blindness, or Guillain-Barré Syndrome. However, this is not an easy task, considering that approximately 80% of ZIKV infection cases are asymptomatic or oligosymptomatic, there are diverse modes of transmission (vertical transmission is through vectors and horizontal transmission through blood, saliva, semen, and urine from infected people), and the fact that ZIKV has a high identity percentage with other cocirculating Flaviviruses such as dengue. Here, we review ZIKV diagnostic methods, with special emphasis on the development of point-of-care diagnostic assays, since these devices commonly have two important advantages: they provide prompt screening and are affordable.