Black swan events in infectious disease describe rare but devastatingly large outbreaks. While experts are skeptical that such events are predictable, it might be possible to identify the warning ...signs of a black swan event. Specifically,
the initiation of an outbreak, key differentiating features could serve as alerts. Such features could be derived from meta-analyses of large outbreaks for multiple infectious diseases. We hypothesized there may be common features among the pathogen, environment, and host epidemiological triad that characterize an infectious disease black swan event. Using Los Alamos National Laboratory's tool, Analytics for Investigation of Disease Outbreaks, we investigated historical disease outbreak information and anomalous events for several infectious diseases. By studying 32 different infectious diseases and global outbreaks, we observed that in the past 20-30 years, there have been potential black swan events in the majority of infectious diseases analyzed. Importantly, these potential black swan events cannot be attributed to the first introduction of the disease to a susceptible host population. This paper describes our observations and perspectives and illustrates the value of broad analysis of data across the infectious disease realm, providing insights that may not be possible when we focus on singular infectious agents or diseases. Data analytics could be developed to warn health authorities at the beginning of an outbreak of an impending black swan event. Such tools could complement traditional epidemiological modeling to help forecast future large outbreaks and facilitate timely warning and effective, targeted resource allocation for mitigation efforts.
Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and ...recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of
Yersinia pestis
. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical.
A novel multidimensional scan technique combining MS
1
and MS/MS scans greatly enhances bacterial differentiation from low resolution data for clinical and field-forward applications.
Targeted alpha therapy offers unique opportunities for the treatment of tumours and infections. Here, we report the development of a new radioimmunoconjugate construct that targets SARS-CoV-2 ...infected cells, which act as viral reservoirs and promote virus replication and infection spread. The chosen antibody selectively binds to the ACE2-receptor binding domain of the spike protein, and prevents the protein binding to the receptor. Furthermore, the antibody has been radiolabelled with
225
Ac, and the therapeutic performance of the resulting radioimmunoconjugate has been demonstrated
in vitro
against cells mimicking SARS-CoV-2 infection.
Cytotoxicity was induced in cells mimicking SARS-CoV-2 infection by a
225
Ac-radiolabeled antibody that targets the RBD2 of the spike protein.
Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic ...resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (alphaF1Ig AM2 and alphaF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that alphaF1Ig AM2 and alphaF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 mug of IgGs alphaF1Ig AM2 and alphaF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of alphaF1Ig AM2 and alphaF1Ig AM8 and support their development as potential medical countermeasures against plague.
Autophagy is a critical mechanism deployed by eukaryotic cells in response to stress, including viral infection, to boost the innate antimicrobial responses. However, an increasing number of ...pathogens hijack the autophagic machinery to facilitate their own replication. Influenza A virus (IAV), responsible for several global pandemics, has an intricate dependence on autophagy for successful replication in mammalian cells. To elucidate key chokepoints in the host stress responses facilitating IAV replication, we constructed a meta‐transcriptome of IAV and host gene expression dynamics during early (1–3 hpi), mid (4–6 hpi), and late (8–12 hpi) stages of the viral replication cycle at two multiplicities of infection (MOI): 1 and 5. We supplemented the global transcriptome study with phosphoproteomic analysis of stress‐activated protein kinase (SAPK/JNK) signaling in lung carcinoma (predominantly used as an in vitro model of IAV replication) and normal human bronchial epithelial cells. We report significant differences in the activation profiles of autophagy regulating genes upon IAV infection at the two MOI as well as divergent dependence on ULK1 signaling within the normal and cancer cells. Regardless of the cell model, JNK‐Thr187 signaling was crucial for the production of infectious viral particles.
Genes regulating mTORC1 and ULK1 signaling pathways are simultaneously activated in Influenza A‐infected lung bronchial epithelium cells. The virus load significantly affects the gene expression dynamics of mTORC1. Stress‐activated protein kinase (SAPK/JNK) signaling is triggered upon accumulation of high levels of viral proteins and coincides with the activation of autophagy.
Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing ...functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras. Here, we describe a high throughput method based on yeast display to directly select antibodies most suitable for conversion to fluorescent chimera. A library of scFv binders was converted to a fluorescent chimeric form, by cloning thermal green protein into the linker between VH and VL, and directly selecting for both binding and fluorescent functionality. This allowed us to directly identify antibodies functional in the single chain TGP format, that manifest higher protein expression, easier protein purification, and one-step binding assays.
Abstract
In vitro display technologies based on phage and yeast have a successful history of selecting single-chain variable fragment (scFv) antibodies against various targets. However, single-chain ...antibodies are often unstable and poorly expressed in Escherichia coli. Here, we explore the feasibility of converting scFv antibodies to an intrinsically fluorescent format by inserting the monomeric, stable fluorescent protein named thermal green, between the light- and heavy-chain variable regions. Our results show that the scTGP format maintains the affinity and specificity of the antibodies, improves expression levels, allows one-step fluorescent assay for detection of binding and is a suitable reagent for epitope binning. We also report the crystal structure of an scTGP construct that recognizes phosphorylated tyrosine on FcεR1 receptor of the allergy pathway.
Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids ...(e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the specific protein prior to analysis and do not identify the specific residue that has been modified (in the case that multiple amino acids have been modified). Phosphorylation state-specific antibodies (PSSAs) developed to recognize post-translational modifications within a specific amino acid sequence can be used to study the timeline of modifications during a signal cascade. We used the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We selected three β-subunit antibodies that recognized: 1) phosphorylation of tyrosines Y
or Y
; 2) phosphorylation of the Y
tyrosine; and 3) phosphorylation of all three tyrosines. We used these antibodies to study the receptor activation timeline of FcεR1 in rat basophilic leukemia cells (RBL-2H3) upon stimulation with DNP
-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y
) of the receptor and applied this antibody to evaluate receptor activation. Recognition patterns of these antibodies show different timelines for phosphorylation of tyrosines in both β and γ subunits. Our methodology provides a strategy to select antibodies specific to post-translational modifications and provides new reagents to study mast cell activation by the high-affinity IgE receptor, FcεRI.
Interactions between the cytoplasmic domains of viral transmembrane proteins and host machinery often determine the outcome of viral infection. The M2 protein of influenza A has been identified as a ...key player in autophagy-mediated viral replication. Here, we describe the engineering and validation of an antibody specific for the cytoplasmic domain of the M2 protein. Through phage and yeast display selection techniques, we obtained an antibody that recognizes: 1) the M2 cytoplasmic domain purified from bacterial inclusion bodies and refolded, 2) full-length M2 recombinant protein expressed in mammalian cells, and 3) native M2 protein in influenza A infected cells. This antibody can serve as a molecular tool to enhance our knowledge of protein-protein interactions between influenza A virus and the host cell machinery. We anticipate the methods described herein will further the development of antibodies specific to the cytoplasmic domains of transmembrane proteins.
Accessible epidemiological data are of great value for emergency preparedness and response, understanding disease progression through a population, and building statistical and mechanistic disease ...models that enable forecasting. The status quo, however, renders acquiring and using such data difficult in practice. In many cases, a primary way of obtaining epidemiological data is through the internet, but the methods by which the data are presented to the public often differ drastically among institutions. As a result, there is a strong need for better data sharing practices. This paper identifies, in detail and with examples, the three key challenges one encounters when attempting to acquire and use epidemiological data: (1)
, (2)
, and (3)
. These challenges are used to provide suggestions and guidance for improvement as these systems evolve in the future. If these suggested data and interface recommendations were adhered to, epidemiological and public health analysis, modeling, and informatics work would be significantly streamlined, which can in turn yield better public health decision-making capabilities.
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