Acquired insights from the long-term surveillance of SARS-CoV-2 RNA for COVID-19 monitoring: The case of Monterrey Metropolitan Area (Mexico).

Wastewater-based epidemiology offers a time- and cost-effective way to monitor SARS-CoV-2 spread in communities and therefore represents a complement to clinical testing. WBE applicability has been demonstrated in a number of cases over short-term periods as a method for tracking the prevalence of SARS-CoV-2 and an early-warning tool for predicting outbreaks in the population. This study reports SARS-CoV-2 viral loads from wastewater treatment plants (WWTPs) and hospitals over a 6-month period (June to December 2020). Results show that the overall range of viral load in positive tested samples was between 1.2 × 103 and 3.5 × 106 gene copies/l, unveiling that secondary-treated wastewaters mirrored the viral load of influents. The interpretation suggests that the viral titers found in three out of four WWTPs were associated to clinical COVID-19 surveillance indicators preceding 2-7 days the rise of reported clinical cases. The median wastewater detection rate of SARS-CoV-2 was one out of 14,300 reported new cases. Preliminary model estimates of prevalence ranged from 0.02 to 4.6% for the studied period. This comprehensive statistical and epidemiological analysis demonstrates that the applied wastewater-based approach to COVID-19 surveillance is in general consistent and feasible, although there is room for improvements.

nurP28, a New-to-Nature Zein-Derived Peptide, Enhances the Therapeutic Effect of Docetaxel in Breast Cancer Monolayers and Spheroids.

The development of novel cancer therapeutic strategies has garnered increasing interest in cancer research. Among the therapeutic choices, chemosensitizers have shown exciting prospects. Peptides are an attractive alternative among the molecules that may be used as chemosensitizers. We rationally designed a new-to-nature peptide, nurP28, derived from the 22-kDa α-zein protein sequence (entry Q00919_MAIZE). The resultant sequence of the nurP28 peptide after the addition of arginine residues was LALLALLRLRRRATTAFIIP, and we added acetyl and amide groups at the N- and C-terminus, respectively, for capping. We evaluated the cytotoxicity of the nurP28 peptide alone and in combination with docetaxel in fibroblast monolayers and breast cancer monolayers and spheroids. Our results indicated that nurP28 is not cytotoxic to human fibroblasts or cancer cells. Nevertheless, when combined with 1 µM docetaxel, 3 ng/mL nurP28 induced equivalent (in MCF7 monolayers) and higher (in MCF7 spheroids) cytotoxic effects than 10-fold higher doses of docetaxel alone. These findings suggest that nurP28 may act as a chemosensitizer in breast cancer treatment. This study describes the enhancing "anti-cancer" effects of nurP28 in breast cancer 2D and 3D cultures treated with docetaxel. Further studies should explore the mechanisms underlying these effects and assess the clinical potential of our findings using animal models.

Strategies for antimicrobial peptide coatings on medical devices: a review and regulatory science perspective.

Indwelling and implanted medical devices are subject to contamination by microbial pathogens during surgery, insertion or injection, and ongoing use, often resulting in severe nosocomial infections. Antimicrobial peptides (AMPs) offer a promising alternative to conventional antibiotics to reduce the incidence of such infections, as they exhibit broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria, microbial biofilms, fungi, and viruses. In this review-perspective, we first provide an overview of the progress made in this field over the past decade with an emphasis on the local release of AMPs from implant surfaces and immobilization strategies for incorporating these agents into a wide range of medical device materials. We then provide a regulatory science perspective addressing the characterization and testing of AMP coatings based on the type of immobilization strategy used with a focus on the US market regulatory niche. Our goal is to help narrow the gulf between academic studies and preclinical testing, as well as to support a future literature base in order to develop the regulatory science of antimicrobial coatings.

Potential gingival crevicular fluid and serum biomarkers by stage of HIV infection.

OBJECTIVE: This study evaluates the potential of gingival crevicular fluid and serum cytokines as HIV stage biomarkers. METHODS: Gingival crevicular fluid (GCF) and serum samples from 78 HIV-positive adult male subjects (cases) and 39 HIV-negative male subjects (controls) from Mexico were examined for 17 cytokines using multiplex ELISA. Participants were divided into five subgroups by HIV stage of infection on age-specific CD4+ T-lymphocyte count and antiretroviral therapy (ART), and further correlated to the cytokine levels. RESULTS: GCF concentrations of IL-6, IL-7, IL-10, IL-12, G-CSF and MCP-1, as well as serum concentrations of IL-1ß, IL-2 and IL-6 showed a statistically significant difference among subgroups. We found a significant effect size correlation on cytokines expression levels. Subjects who were not in ART showed significantly higher levels of some of the analyzed cytokines compared to the rest. We found that GCF IL-8 was a significant predictor for the Non-ART HIV status (p<0.05). We observed the same result for GCF G-CSF in the ART Short-term group and serum GM-CSF in the ART Long-term subgroup. CONCLUSION: Results indicate a high variability of GCF and serum cytokines concentrations and low frequency of their detection in different HIV/ART stages. However, within the limits of the present study, some GCF and serum cytokine concentrations correlate positively. Oral and periodontal innate immunity is affected by HIV viremia and ART. GCF IL-8, G-CSF, as well as serum IL-8, MCP-1 and GM-CSF may be useful biomarkers for the detection of disease presence and/or its severity due to HIV infection and ART use.

Spatially and Temporally Controlled Hydrogels for Tissue Engineering.

Recent years have seen tremendous advances in the field of hydrogel-based biomaterials. One of the most prominent revolutions in this field has been the integration of elements or techniques that enable spatial and temporal control over hydrogels' properties and functions. Here, we critically review the emerging progress of spatiotemporal control over biomaterial properties towards the development of functional engineered tissue constructs. Specifically, we will highlight the main advances in the spatial control of biomaterials, such as surface modification, microfabrication, photo-patterning, and three-dimensional (3D) bioprinting, as well as advances in the temporal control of biomaterials, such as controlled release of molecules, photocleaving of proteins, and controlled hydrogel degradation. We believe that the development and integration of these techniques will drive the engineering of next-generation engineered tissues.

Anti-Ebola therapies based on monoclonal antibodies: current state and challenges ahead.

The 2014 Ebola outbreak, the largest recorded, took us largely unprepared, with no available vaccine or specific treatment. In this context, the World Health Organization declared that the humanitarian use of experimental therapies against Ebola Virus (EBOV) is ethical. In particular, an experimental treatment consisting of a cocktail of three monoclonal antibodies (mAbs) produced in tobacco plants and specifically directed to the EBOV glycoprotein (GP) was tested in humans, apparently with good results. Several mAbs with high affinity to the GP have been described. This review discusses our current knowledge on this topic. Particular emphasis is devoted to those mAbs that have been assayed in animal models or humans as possible therapies against Ebola. Engineering aspects and challenges for the production of anti-Ebola mAbs are also briefly discussed; current platforms for the design and production of full-length mAbs are cumbersome and costly.

Evaluation of changes in promoters, use of UCOES and chain order to improve the antibody production in CHO cells.

Therapy with biopharmaceuticals, mainly recombinant antibodies, offers patients higher life expectancy and better life quality than pharmacologic therapy. Countries with the highest scientific development are investing in this kind of therapy, and this is why the optimization of the production of these recombinant proteins would lead to their higher production and lower costs of the final product. Modifications in the use of promoters, the use of recombination regions, and the change in the order of the chains, are some of the genetic engineering changes that can increase the production of recombinant antibodies. In this work, three different promoters were tested: Prom A, hCMV, and EF1-a, for two different antibodies, one anti-TNFa and one anti-CD20+. Changes were made in the order of the chains H-L or L-H and one or two UCOE (ubiquitous chromatin opening element) sequences were also used to identify the combinations that provide the best transient and stable expression for the antibodies in the CHO-s cells. In our results, we observed that the use of the two UCOE regions, with L-H order is almost three times better for the expression of the two different antibodies, while the strength of the promoter is conditioned by the sequence of each expressed protein.

Proteomic-based biomarker discovery for development of next generation diagnostics.

In the post-genome age, proteomics is receiving significant attention because they provide an invaluable source of biological structures and functions at the protein level. The search for disease-specific biomarkers for diagnostic and/or therapeutic applications is one of the areas that proteomics is having a significant impact. Thus, the identification of a "good" biomarker enables a more accurate early diagnosis and prognosis of disease. Rapid advancements in mass spectrometry (MS) instrumentation, liquid chromatography MS (LCMS), protein microarray technology, and other protein profiling methodologies have a substantial expansion of our toolbox to identify disease-specific protein and peptide biomarkers. This review covers a selection of widely used proteomic technologies for biomarker discovery. In addition, we describe the most commonly used approaches for diagnosis based on proteomic biomarkers and further discuss trends and critical challenges during development of cost-effective rapid diagnostic tests and microfluidic diagnostic systems based on proteomic biomarkers.

Self-Diagnosis of SARS-CoV-2 from Saliva Samples at Home: Isothermal Amplification Enabled by Do-It-Yourself Portable Incubators and Laminated Poly-ethyl Sulfonate Membranes.

COVID-19 made explicit the need for rethinking the way in which we conduct testing for epidemic emergencies. During the COVID-19 pandemic, the dependence on centralized lab facilities and resource-intensive methodologies (e.g., RT-qPCR methods) greatly limited the deployment of widespread testing efforts in many developed and underdeveloped countries. Here, we illustrate the development of a simple and portable diagnostic kit that enables self-diagnosis of COVID-19 at home from saliva samples. We describe the development of a do-it-yourself (DIY) incubator for Eppendorf tubes that can be used to conduct SARS-CoV-2 detection with competitive sensitivity and selectivity from saliva at home. In a proof-of-concept experiment, we assembled Eppendorf-tube incubators at our home shop, prepared a single-tube mix of reagents and LAMP primers in our lab, and deployed these COVID-19 detection kits using urban delivery systems (i.e., Rappifavor or Uber) to more than 15 different locations in Monterrey, México. This straightforward strategy enabled rapid and cost-effective at-home molecular diagnostics of SARS-CoV-2 from real saliva samples with a high sensitivity (100%) and high selectivity (87%).

A 3D-printed tumor-on-chip: user-friendly platform for the culture of breast cancer spheroids and the evaluation of anti-cancer drugs.

Tumor-on-chips (ToCs) are useful platforms for studying the physiology of tumors and evaluating the efficacy and toxicity of anti-cancer drugs. However, the design and fabrication of a TOC system is not a trivial venture. We introduce a user-friendly, flexible, 3D-printed microfluidic device that can be used to culture cancer cells or cancer-derived spheroids embedded in hydrogels under well-controlled environments. The system consists of two lateral flow compartments (left and right sides), each with two inlets and two outlets to deliver cell culture media as continuous liquid streams. The central compartment was designed to host a hydrogel in which cells and microtissues can be confined and cultured. We performed tracer experiments with colored inks and 40-kDa fluorescein isothiocyanate dextran to characterize the transport/mixing performances of the system. We also cultured homotypic (MCF7) and heterotypic (MCF7-BJ) spheroids embedded in gelatin methacryloyl hydrogels to illustrate the use of this microfluidic device in sustaining long-term micro-tissue culture experiments. We further demonstrated the use of this platform in anticancer drug testing by continuous perfusion of doxorubicin, a commonly used anti-cancer drug for breast cancer. In these experiments, we evaluated drug transport, viability, glucose consumption, cell death (apoptosis), and cytotoxicity. In summary, we introduce a robust and friendly ToC system capable of recapitulating relevant aspects of the tumor microenvironment for the study of cancer physiology, anti-cancer drug transport, efficacy, and safety. We anticipate that this flexible 3D-printed microfluidic device may facilitate cancer research and the development and screening of strategies for personalized medicine. .

Placing biofabrication into the context of human disease modeling.

The field of biofabrication has seen tremendous advances in the past decade. More recently, the emerging role of biofabrication in allowing faithful generation of models of human tissues in their healthy and diseased states has been demonstrated and has rapidly expanded. These biomimetic models are potentially widely applicable in a range of research and translational areas including but not limited to fundamental biology studies as well as screening of chemical compounds, such as therapeutic agents. The United States Food and Drug Administration Modernization Act 2.0, which now no longer requires animal tests before approving human drug trials, will likely further boost the field in the years to come. This Special Issue, with a collection of 11 excellent research articles, thus focuses on showcasing the latest developments of biofabrication towards human disease modeling, spanning from 3D (bo)printing to organ-on-a-chip as well as their integration.

Structural and biological engineering of 3D hydrogels for wound healing.

Chronic wounds have become one of the most important issues for healthcare systems and are a leading cause of death worldwide. Wound dressings are necessary to facilitate wound treatment. Engineering wound dressings may substantially reduce healing time, reduce the risk of recurrent infections, and reduce the disability and costs associated. In the path of engineering of an ideal wound dressing, hydrogels have played a leading role. Hydrogels are 3D hydrophilic polymeric structures that can provide a protective barrier, mimic the native extracellular matrix (ECM), and provide a humid environment. Due to their advantages, hydrogels (with different architectural, physical, mechanical, and biological properties) have been extensively explored as wound dressing platforms. Here we describe recent studies on hydrogels for wound healing applications with a strong focus on the interplay between the fabrication method used and the architectural, mechanical, and biological performance achieved. Moreover, we review different categories of additives which can enhance wound regeneration using 3D hydrogel dressings. Hydrogel engineering for wound healing applications promises the generation of smart solutions to solve this pressing problem, enabling key functionalities such as bacterial growth inhibition, enhanced re-epithelialization, vascularization, improved recovery of the tissue functionality, and overall, accelerated and effective wound healing.

3D-Printed Tumor-on-Chip for the Culture of Colorectal Cancer Microspheres: Mass Transport Characterization and Anti-Cancer Drug Assays.

Tumor-on-chips have become an effective resource in cancer research. However, their widespread use remains limited due to issues related to their practicality in fabrication and use. To address some of these limitations, we introduce a 3D-printed chip, which is large enough to host ~1 cm3 of tissue and fosters well-mixed conditions in the liquid niche, while still enabling the formation of the concentration profiles that occur in real tissues due to diffusive transport. We compared the mass transport performance in its rhomboidal culture chamber when empty, when filled with GelMA/alginate hydrogel microbeads, or when occupied with a monolithic piece of hydrogel with a central channel, allowing communication between the inlet and outlet. We show that our chip filled with hydrogel microspheres in the culture chamber promotes adequate mixing and enhanced distribution of culture media. In proof-of-concept pharmacological assays, we biofabricated hydrogel microspheres containing embedded Caco2 cells, which developed into microtumors. Microtumors cultured in the device developed throughout the 10-day culture showing >75% of viability. Microtumors subjected to 5-fluorouracil treatment displayed <20% cell survival and lower VEGF-A and E-cadherin expression than untreated controls. Overall, our tumor-on-chip device proved suitable for studying cancer biology and performing drug response assays.

The receptor-binding domain of influenza virus hemagglutinin produced in Escherichia coli folds into its native, immunogenic structure.

The hemagglutinin (HA) surface glycoprotein promotes influenza virus entry and is the key protective antigen in natural immunity and vaccines. The HA protein is a trimeric envelope glycoprotein consisting of a globular receptor-binding domain (HA-RBD) that is inserted into a membrane fusion-mediating stalk domain. Similar to other class I viral fusion proteins, the fusogenic stalk domain spontaneously refolds into its postfusion conformation when expressed in isolation, consistent with this domain being trapped in a metastable conformation. Using X-ray crystallography, we show that the influenza virus HA-RBD refolds spontaneously into its native, immunogenic structure even when expressed in an unglycosylated form in Escherichia coli. In the 2.10-Å structure of the HA-RBD, the receptor-binding pocket is intact and its conformational epitopes are preserved. Recombinant HA-RBD is immunogenic and protective in ferrets, and the protein also binds with specificity to sera from influenza virus-infected humans. Overall, the data provide a structural basis for the rapid production of influenza vaccines in E. coli. From an evolutionary standpoint, the ability of the HA-RBD to refold spontaneously into its native conformation suggests that influenza virus acquired this domain as an insertion into an ancestral membrane-fusion domain. The insertion of independently folding domains into fusogenic stalk domains may be a common feature of class I viral fusion proteins.

Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies.

The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×103 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×103 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×103 and 11×103 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages. We anticipate that the cost-effective and easily implemented protocols presented here will greatly facilitate the generation of EBs, thereby further democratizing the worldwide ability to conduct organoid-based research.

Neuron(s)-on-a-chip: A review of the design and use of microfluidic systems for neural tissue culture.

Neuron-on-chip (NoC) systems-microfluidic devices in which neurons are cultured-have become a promising alternative to replace or minimize the use of animal models and have greatly facilitated in vitro research. Here, we review and discuss current developments in neuron-on-chip platforms, with a particular emphasis on existing biological models, culturing techniques, biomaterials, and topologies. We also discuss how the architecture, flow, and gradients affect neuronal growth, differentiation, and development. Finally, we discuss some of the most recent applications of NoCs in fundamental research (i.e., studies on the effects of electrical, mechanical/topological, or chemical stimuli) and in disease modeling.

Culture of cancer spheroids and evaluation of anti-cancer drugs in 3D-printed miniaturized continuous stirred tank reactors (mCSTRs).

Cancer continues to be a leading cause of mortality in modern societies; therefore, improved and more reliablein vitrocancer models are needed to expedite fundamental research and anti-cancer drug development. Here, we describe the use of a miniaturized continuous stirred tank reactor (mCSTR) to first fabricate and mature cancer spheroids (i.e. derived from MCF7 cells, DU145 cells, and a mix of MCF7 cells and fibroblasts), and then to conduct anti-cancer drug assays under continuous perfusion. This 3 ml mCSTR features an off-center agitation system that enables homogeneous chaotic laminar mixing at low speeds to support cell aggregation. We incubated cell suspensions for 3 d in ultra-low-attachment plates to allow formation of discoid cell aggregates (∼600µm in diameter). These cell aggregates were then transferred into mCSTRs and continuously fed with culture medium. We characterized the spheroid morphology and the expression of relevant tumor biomarkers at different maturation times for up to 4 weeks. The spheroids progressively increased in size during the first 5-6 d of culture to reach a steady diameter between 600 and 800µm. In proof-of-principle experiments, we demonstrated the use of this mCSTR in anti-cancer drug testing. Three drugs commonly used in breast cancer treatment (doxorubicin, docetaxel, and paclitaxel) were probed at different concentrations in MCF7-derived spheroids. In these experiments, we evaluated cell viability, glucose consumption, spheroid morphology, lactate dehydrogenase activity, and the expression of genes associated with drug resistance (ABCB1andABCC1) and anti-apoptosis (Bcl2). We envision the use of this agitated system as a tumor-on-a-chip platform to expedite efficacy and safety testing of novel anti-cancer drugs and possibly in personalized medicine applications.

Low-Cost Light-Based GelMA 3D Bioprinting via Retrofitting: Manufacturability Test and Cell Culture Assessment.

Light-based bioprinter manufacturing technology is still prohibitively expensive for organizations that rely on accessing three-dimensional biological constructs for research and tissue engineering endeavors. Currently, most of the bioprinting systems are based on commercial-grade-based systems or modified DIY (do it yourself) extrusion apparatuses. However, to date, few examples of the adoption of low-cost equipment have been found for light-based bioprinters. The requirement of large volumes of bioinks, their associated cost, and the lack of information regarding the parameter selection have undermined the adoption of this technology. This paper showcases the retrofitting and assessing of a low-cost Light-Based 3D printing system for tissue engineering. To evaluate the potential of a proposed design, a manufacturability test for different features, machine parameters, and Gelatin Methacryloyl (GelMA) concentrations for 7.5% and 10% was performed. Furthermore, a case study of a previously seeded hydrogel with C2C12 cells was successfully implemented as a proof of concept. On the manufacturability test, deviational errors were found between 0.7% to 13.3% for layer exposure times of 15 and 20 s. Live/Dead and Actin-Dapi fluorescence assays after 5 days of culture showed promising results in the cell viability, elongation, and alignment of 3D bioprinted structures. The retrofitting of low-cost equipment has the potential to enable researchers to create high-resolution structures and three-dimensional in vitro models.

One-Step Bioprinting of Multi-Channel Hydrogel Filaments Using Chaotic Advection: Fabrication of Pre-Vascularized Muscle-Like Tissues.

The biofabrication of living constructs containing hollow channels is critical for manufacturing thick tissues. However, current technologies are limited in their effectiveness in the fabrication of channels with diameters smaller than hundreds of micrometers. It is demonstrated that the co-extrusion of cell-laden hydrogels and sacrificial materials through printheads containing Kenics static mixing elements enables the continuous and one-step fabrication of thin hydrogel filaments (1 mm in diameter) containing dozens of hollow microchannels with widths as small as a single cell. Pre-vascularized skeletal muscle-like filaments are bioprinted by loading murine myoblasts (C2C12 cells) in gelatin methacryloyl - alginate hydrogels and using hydroxyethyl cellulose as a sacrificial material. Higher viability and metabolic activity are observed in filaments with hollow multi-channels than in solid constructs. The presence of hollow channels promotes the expression of Ki67 (a proliferation biomarker), mitigates the expression of hypoxia-inducible factor 1-alpha , and markedly enhances cell alignment (i.e., 82% of muscle myofibrils aligned (in ±10°) to the main direction of the microchannels after seven days of culture). The emergence of sarcomeric α-actin is verified through immunofluorescence and gene expression. Overall, this work presents an effective and practical tool for the fabrication of pre-vascularized engineered tissues.

Gelatin-methacryloyl hydrogels containing turnip mosaic virus for fabrication of nanostructured materials for tissue engineering.

Current tissue engineering techniques frequently rely on hydrogels to support cell growth, as these materials strongly mimic the extracellular matrix. However, hydrogels often need ad hoc customization to generate specific tissue constructs. One popular strategy for hydrogel functionalization is to add nanoparticles to them. Here, we present a plant viral nanoparticle the turnip mosaic virus (TuMV), as a promising additive for gelatin methacryloyl (GelMA) hydrogels for the engineering of mammalian tissues. TuMV is a flexuous, elongated, tubular protein nanoparticle (700-750 nm long and 12-15 nm wide) and is incapable of infecting mammalian cells. These flexuous nanoparticles spontaneously form entangled nanomeshes in aqueous environments, and we hypothesized that this nanomesh structure could serve as a nanoscaffold for cells. Human fibroblasts loaded into GelMA-TuMV hydrogels exhibited similar metabolic activity to that of cells loaded in pristine GelMA hydrogels. However, cells cultured in GelMA-TuMV formed clusters and assumed an elongated morphology in contrast to the homogeneous and confluent cultures seen on GelMA surfaces, suggesting that the nanoscaffold material per se did not favor cell adhesion. We also covalently conjugated TuMV particles with epidermal growth factor (EGF) using a straightforward reaction scheme based on a Staudinger reaction. BJ cells cultured on the functionalized scaffolds increased their confluency by approximately 30% compared to growth with unconjugated EGF. We also provide examples of the use of GelMA-TuMV hydrogels in different biofabrication scenarios, include casting, flow-based-manufacture of filaments, and bioprinting. We envision TuMV as a versatile nanobiomaterial that can be useful for tissue engineering.