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Prothymosin α (ProTα), a polypeptide containing the sequence of thymosin α1 (Tα1) at its NH2-terminus, has been isolated from calf thymocytes in a concentration in the order of that found in the whole thymic gland. As deduced from the analysis of their tryptic peptides, calf ProT‘ differs from the rat polypeptide at least in an alanine residue replacing valine at position 92. Thymocytes cultured in a radioactive medium exhibit an important secretory activity. ProTα being one of the products synthesized and exported to the culture medium. Large and small thymocytc subpopulations front calf and rat differ in their capacity to synthesize ProTα. The polypeptide is produced in a major concentration by large thymocytes. However, all the calf and rat thymocytc subpopulations show a similar capacity to secrete ProTα, the amount of the newly synthesized polypeptide recovered from cell culture supernatants being 80–90% of that found in thymocytc extracts. [ABSTRACT FROM AUTHOR]

Biosensors have been used for a remarkable array of applications, including infectious diseases, environmental monitoring, cancer diagnosis, food safety, and numerous others. In particular, the global COVID-19 pandemic has exposed a need for rapid tests, so the type of biosensor that has gained considerable interest recently are immunoassays, which are used for rapid diagnostics. The performance of paper-based lateral flow and dipstick immunoassays is influenced by the physical properties of the nanoparticles (NPs), NP-antibody conjugates, and paper substrate. Many materials innovations have enhanced diagnostics by increasing sensitivity or enabling unique readouts. However, negative side effects can arise at the interface between the biological sample and biomolecules and the NP or paper substrate, such as non-specific adsorption and protein denaturation. In this Perspective, we discuss the immunoassay components and highlight chemistry and materials innovations that can improve sensitivity. We also explore the range of bio-interface issues that can present challenges for immunoassays.

The COVID-19 pandemic has led to an unprecedented global health challenge, creating sudden, massive demands for diagnostic testing, treatment, therapies, and vaccines. In particular, the development of diagnostic assays for SARS-CoV-2 has been pursued as they are needed for quarantine, disease surveillance, and patient treatment. One of the major lessons the pandemic highlighted was the need for fast, cheap, scalable and reliable diagnostic methods, such as paper-based assays. Furthermore, it has previously been suggested that paper-based tests may be more suitable for settings with lower resource availability and may help alleviate some supply chain challenges which arose during the COVID-19 pandemic. Therefore, we explore how such devices may fit in a comprehensive diagnostic strategy and how some of the challenges to the technology, e.g. low sensitivity, may be addressed. We discuss the properties of the SARS-CoV-2 virus itself, the COVID-19 disease pathway, and the immune response. We then describe the different diagnostic strategies that have been pursued, focusing on molecular strategies for viral genetic material, antigen tests, and serological assays, and innovations for improving the diagnostic sensitivity and capabilities. Finally, we discuss pressing issues for the future, and what needs to be addressed for the ongoing pandemic and future outbreaks.

COVID-19 first appeared in December of 2019 in Wuhan, China. Since then, it has become a global pandemic. A robust and scalable diagnostics strategy is crucial for containing and monitoring the pandemic. RT-PCR is a known, reliable method for COVID-19 diagnostics, which can differentiate between SARS-CoV-2 and other viruses. However, PCR is location-dependent, time-consuming, and relatively expensive. Thus, there is a need for a more flexible method, which may be produced in an off-the-shelf format and distributed more widely. Paper-based immunoassays can fulfill this function. Here, we present the first steps toward a paper-based test, which can differentiate between different spike proteins of various coronaviruses, SARS-CoV-1, SARS-CoV-2, and CoV-HKU1, with negligible cross-reactivity for HCoV-OC43 and HCoV-229E in a single assay, which takes less than 30 min. Furthermore, our test can distinguish between fractions of the same spike protein. This is done by an altered assay design with four test line locations where each antigen builds a unique, identifiable binding pattern. The effect of several factors, such as running media, immunoprobe concentration, and antigen interference, is considered. We find that running media has a significant effect on the final binding pattern where human saliva provides results while human serum leads to the lowest signal quality.

There has been an increasing and urgent demand to develop nucleic acid bioassays which not only offer high analytical performance but which are also amenable with point-of-care testing. Hydrogels present a versatile class of materials with biocompatible antifouling properties and the ability to be engineered for a range of advanced sensing applications. Fibrous substrates like nitrocellulose offer low-cost and durable platforms to run complex bioassays while enabling portability and ease of handling. We demonstrate herein the ability to synergistically combine these two materials into a portable biosensing platform by leveraging projection lithography. We demonstrate the direct polymerization of hydrogel sensing motifs within a range of fibrous substrates with precise control over their shape, size, location, and functionality. Spatial encoding of the hydrogel motifs enables the multiplex detection of multiple biomarkers on the same test. As a proof-of-concept, we apply the platform to the detection of microRNA, an emerging class of circulating biomarkers with promising potential for early diagnosis and monitoring of cancer. The assay offers a large dynamic range (over three orders of magnitude), high sensitivity (limit of detection of 2.5 amol), as well as versatility and ease of handling. Finally, the bioassay is validated using real biological samples, namely, total RNA extracted from the sera of late-stage breast cancer patients, demonstrating its utility and compatibility with clinical biosensing applications.

Nucleic acid amplification tests (NAATs), which amplify and detect pathogen nucleic acids, are vital methods to diagnose diseases, particularly in cases where patients exhibit low levels of infection. For many blood-borne pathogens such as HIV or Plasmodium falciparum, it is necessary to first extract pathogen RNA or DNA from patient blood prior to NAAT analysis. Traditional nucleic acid extraction methods are expensive, resource-intensive and are often difficult to deploy to resource-limited areas where many blood-borne infections are widespread. Here, we describe a portable, paper-and-plastic device, called SNAPflex, for instrument-free nucleic acid extraction from whole blood, which builds upon our previous work for RNA extraction using a pressure-driven extraction system. SNAPflex shows improved HIV RNA extraction from simulated patient samples compared to traditional extraction methods as well as long-term stability of extracted RNA without the need for cold storage. We further demonstrated successful extraction and recovery of P. falciparum DNA from cultured parasites in whole blood. SNAPflex was designed to be easily manufacturable and deployable to resource-limited settings.

We exploit the cross-reactivity of dengue (DENV) and Zika (ZIKV) virus polyclonal antibodies for nonstructural protein 1 (NS1) to construct a selective sensor that can detect yellow fever virus (YFV) NS1 in a manner similar to chemical olfaction. DENV and ZIKV antibodies were screened for their ability to bind to DENV, ZIKV, and YFV NS1 by enzyme linked immunosorbent assay (ELISA) and in pairs in paper immunoassays. A strategic arrangement of antibodies immobilized on paper and conjugated to different colored gold NPs was used to distinguish the three biomarkers. Machine learning of test area RGB values showed that with two spots, readout accuracies of 100% and 87% were obtained for both pure NS1 and DENV/YFV mixtures, respectively. Additional image preprocessing allowed differentiation between all four DENV serotypes with 92% accuracy. The technique was extended to hack a commercial DENV test to detect YFV and ZIKV by augmentation with DENV and ZIKV polyclonal antibodies.

Historically, many industries such as manufacturing have undergone a trend away from centralized, large-scale production toward a more distributed form. Currently, this same trend is witnessed in biological manufacturing and bioprocessing, with the rise of biological foundries where one can synthesize, grow, isolate, and purify a broad range of biologics. The adoption of distributed practices for biological processing has significant implications for healthcare, diagnostics, and therapies. This essay discusses the many diverse factors that have facilitated this growth, ranging from the establishment of available biological components, or "parts," low-cost programmable hardware, and others. Currently existing examples of distributed biological foundries are also identified, separating the discussion into those that are accessible only by elite users and the more recent emerging foundries that are more accessible to the general population. Taking lessons from other fields, it is argued that this trend toward distributed biological manufacturing is inevitable, so adapting to this trend is important for the progress of creating new therapeutics, sensors, diagnostics, and reagents for biomedical applications., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Paper-based sensors and assays have been highly attractive for numerous biological applications, including rapid diagnostics and assays for disease detection, food safety, and clinical care. In particular, the paper immunoassay has helped drive many applications in global health due to its low cost and simplicity of operation. This review is aimed at examining the fundamentals of the technology, as well as different implementations of paper-based assays and discuss novel strategies for improving their sensitivity, performance, or enabling new capabilities. These innovations can be categorized into using unique nanoparticle materials and structures for detection via different techniques, novel biological species for recognizing biomarkers, or innovative device design and/or architecture.

The design and fabrication of reconfigurable, modular paperfluidics driven by a prefabricated reusable block library, asynchronous modular paperfluidic linear instrument-free (Ampli) block, are reported. The blocks are inspired by the plug-and-play modularity of electronic breadboards that lower prototyping barriers in circuit design. The resulting biochemical breadboard is a paperfluidic construction set that can be functionalized with chemical, biological, and electrical elements. Ampli blocks can form standard paperfluidic devices without any external instrumentation. Furthermore, their modular nature enhances fluidics in ways that fixed devices cannot. The blocks' ability to start, stop, modify, and reverse reaction flows, reagents, and rates in real time is demonstrated. These enhancements allow users to increase colorimetric signals, fine tune reaction times, and counter check multiplexed diagnostics for false positives or negatives. The modular construction demonstrates that field-ready, distributed fabrication of paper analytical systems can be standardized without requiring the "black box" of craft and technique inherent in paper-based systems. Ampli assembly and point-of-care redesign extends the usability of paper analytical systems and invites user-driven prototyping beyond the lab setting demonstrating "Design for Hack" in diagnostics., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Histoplasma duboisii, a variant of Histoplasma capsulatum that causes "African histoplasmosis," can be resistant to itraconazole, requiring intravenous amphotericin B treatment. Rarely, these patients do not respond to intravenous antifungal therapy, and in such cases, patients may progress to develop secondary hemophagocytic lymphohistiocytosis (HLH). We present a case of a 34-year-old male patient with sickle cell disease who presented with a 5-month history of an enlarging painless axillary mass, persistent low grade fevers, night sweats, weight loss, and anorexia. An excisional biopsy of the right axillary lymph node revealed yeast and granulomas consistent with histoplasma infection. He was started on oral itraconazole. After 4 weeks of therapy, laboratory evaluation revealed worsening anemia, thrombocytopenia, and transaminitis. Due to failure of oral therapy, he was admitted for intravenous amphotericin B treatment. During his hospital course anemia, thrombocytopenia, and transaminitis all worsened. A bone marrow biopsy was done that was consistent with HLH. His clinical status continued to deteriorate, developing multiorgan failure and disseminated intravascular coagulation. He unfortunately had a cardiorespiratory arrest after eight days of admission and passed away.

Innovations in international health require the use of state-of-the-art technology to enable clinical chemistry for diagnostics of bodily fluids. We propose the implementation of a portable and affordable lock-in amplifier-based instrument that employs digital technology to perform biochemical diagnostics on blood, urine, and other fluids. The digital instrument is composed of light source and optoelectronic sensor, lock-in detection electronics, microcontroller unit, and user interface components working with either power supply or batteries. The instrument performs lock-in detection provided that three conditions are met. First, the optoelectronic signal of interest needs be encoded in the envelope of an amplitude-modulated waveform. Second, the reference signal required in the demodulation channel has to be frequency and phase locked with respect to the optoelectronic carrier signal. Third, the reference signal should be conditioned appropriately. We present three approaches to condition the signal appropriately: high-pass filtering the reference signal, precise offset tuning the reference level by low-pass filtering, and by using a voltage divider network. We assess the performance of the lock-in instrument by comparing it to a benchmark device and by determining protein concentration with single-color absorption measurements. We validate the concentration values obtained with the proposed instrument using chemical concentration measurements. Finally, we demonstrate that accurate retrieval of phase information can be achieved by using the same instrument.

Prothymosin alpha (ProTalpha) is an abundant mammalian acidic nuclear protein whose expression is related to cell proliferation. Here we report that in HL-60 cells overexpressing ProTalpha, the accessibility of micrococcal nuclease to chromatin is strongly increased. In the DNA ladder generated by the nuclease activity, the sizes of the mononucleosome (146 bp, the DNA fragment that is bound to the histone octamer) and its multimers correspond to nucleosomes lacking histone H1. The percentage of histone-H1-depleted chromatin (active chromatin) is also higher in the cells overexpressing ProTalpha. On the basis of these and previous findings, we propose a biological role for ProTalpha in the remodelling of chromatin fibres through its interaction with histone H1.

Thymus and thymocytes from calf were extracted under isotonic conditions in the presence of protease inhibitors or under severe denaturing conditions (after quick freezing and thawing in boiling 0.1 M NaCl). The extracts, as well as the medium in which the thymocytes were obtained from thymus fragments (thymocyte supernatants), were size-fractionated by ultrafiltration. As in whole thymus isotonic extracts, thymosin alpha 1 [A. L. Goldstein, T. L. K. Low, M. McAdoo, J. McClure, G. B. Thurman, J. Rossio, C-Y. Lai, D. Chang, S-S. Wang, C. Harvey, A. H. Ramel, and J. Meienhofer (1977) Proc. Natl. Acad. Sci. USA 74, 725-729] was contained in isotonic extracts from thymocytes and also in thymocyte supernatants, as determined by isoelectric focusing and reverse-phase HPLC analysis. The extraction under denaturing conditions mainly yielded products with molecular masses over 50,000, showing very similar isoelectric focusing patterns in both thymocytes and whole thymus extracts. As deduced by isoelectric focusing analysis of diverse size-fractionated products, a strong association capacity seems to be responsible for an apparently high molecular mass of the components of these extracts. According to the pI, two of these components were prothymosin alpha [A. A. Haritos, G. J. Goodall, and B. L. Horecker (1984) Proc. Natl. Acad. Sci. USA 81, 1008-1011] and thymosin alpha 1. Prothymosin alpha was not detected in any isotonic extracts or thymocyte supernatants. These data suggest that calf thymocytes are capable of producing thymosin alpha 1, which would arise by natural processing of its precursor.

Prothymosin alpha (ProT alpha), a polypeptide containing the sequence of thymosin alpha 1 (T alpha 1) at its NH2-terminus, has been isolated from calf thymocytes in a concentration in the order of that found in the whole thymic gland. As deduced from the analysis of their tryptic peptides, calf ProT alpha differs from the rat polypeptide at least in an alanine residue replacing valine at position 92. Thymocytes cultured in a radioactive medium exhibit an important secretory activity, ProT alpha being one of the products synthesized and exported to the culture medium. Large and small thymocyte subpopulations from calf and rat differ in their capacity to synthesize ProT alpha. The polypeptide is produced in a major concentration by large thymocytes. However, all the calf and rat thymocyte subpopulations show a similar capacity to secrete ProT alpha, the amount of the newly synthesized polypeptide recovered from cell culture supernatants being 80-90% of that found in thymocyte extracts.