Formamide as the main building block in the origin of nucleic acids

The simplest molecules grouping the four most common elements of the universe H,C,O and N (with the exception of the biologically inert He) are isocyanate HNCO and formamide H2NCOH. Reasons for the availability of formamide on prebiotic Earth are presented. We review evidence showing that formamide in the presence of largely available catalysts and by moderate heating yields the complete set of nucleic bases necessary for the formation of nucleic acids. Formamide also favours the formation of acyclonucleosides and the phosphorylation and trans-phosphorylation of nucleosides, thus providing a plausible chemical frame for the passage from a simple one-carbon compound to nucleic polymers. Physico-chemical conditions exist in which formamide favours the stability of the phosphoester bonds in nucleic polymers relative to that of the same bonds in monomers. Starting from a formamide-laden environment subject only to the laws of chemistry, a hypothesis is outlined sketching the passage towards an aqueous world in which Darwinian rules apply.     

Regions of Diversity 8, 9 and 13 contribute to <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> virulence

Background  

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. Previously, using comparative genomic analyses, 13 regions of genomic plasticity have been identified in the S. pneumoniae genome. These "Regions of Diversity" (RDs) accounted for half the genomic variation observed amongst all pneumococci tested, moreover, were determined to encode a variety of putative virulence factors. To date, genes within 5 RDs have been unequivocally demonstrated to contribute to S. pneumoniae virulence. It is unknown if the remaining RDs also contribute to virulence.     

Transgenic mice expressing a dual, CRE-inducible reporter for the analysis of axon guidance and synaptogenesis

Improved and modular tools are needed for the neuroanatomical dissection of CNS axonal tracts, and to study the cell-intrinsic and cell-extrinsic cues that govern their assembly and plasticity. Here we describe a general purpose transgenic tracer that can be used to visualize axonal tracts and synaptic terminals in any region of the embryonic neural tube or postnatal CNS, on any wild type or mutant genetic background. The construct permits CRE-inducible expression of a dicistronic axonal marker encoding two surface reporter proteins: a farnesylated GFP and the human Placental Alkaline Phosphatase (PLAP). Both proteins localize alongside the neuronal surface, permitting the concomitant detection of cell body, neurites, and presynaptic and postsynaptic sites in the same neuron. This provides a CRE-inducible dual system for imaging neural circuits in vivo, and to study their assembly and remodeling in cultured neurons, neural stem cells, and tissue explants derived from the reporter line. Unlike existing lines, this reporter does not encode a ubiquitously expressed, floxable LacZ gene, permitting the simultaneous analysis of beta galactosidase activity in mutant lines.     

Four disulfide-bridged scorpion beta neurotoxin CssII: heterologous expression and proper folding in vitro

The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and a FXa proteolytic cleavage region. This recombinant vector was transfected into Escherichia coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/l of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7538.6 Da as expected. Since native CssII (nCssII) is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1 Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na(v)1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.     

3D structure of Syk kinase determined by single-particle electron microscopy

The cytoplasmic Syk kinase plays key roles in immune responses and comprises two N-terminal regulatory Src homology 2 (SH2) domains followed by a catalytic region. Atomic structures of these domains have only been solved in isolation. To gain insights into the three-dimensional structure of full-length Syk, we have used single-particle electron microscopy. Syk acquires a closed conformation resembling the inhibited structure of Zap-70, another member of the Syk family. Such configuration suggests an inhibition of the N-terminal domains on its catalytic activity. The phosphotyrosine binding pockets of both SH2 domains are not occluded and they could interact with other phosphoproteins.     

Clinical Data Extraction During Public Health Emergencies: A Blockchain Technology Assessment

Objective:We sought to explore the technical and legal readiness of healthcare institutions for novel data-sharing methods that allow clinical information to be extracted from electronic health records (EHRs) and submitted securely to the Food and Drug Administration''s (FDA''s) blockchain through a secure data broker (SDB).Materials and Methods:This assessment was divided into four sections: an institutional EHR readiness assessment, legal consultation, institutional review board application submission, and a test of healthcare data transmission over a blockchain infrastructure.Results:All participating institutions reported the ability to electronically extract data from EHRs for research. Formal legal agreements were deemed unnecessary to the project but would be needed in future tests of real patient data exchange. Data transmission to the FDA blockchain met the success criteria of data connection from within the four institutions'' firewalls, externally to the FDA blockchain via a SDB.Discussion:The readiness survey indicated advanced analytic capability in hospital institutions and highlighted inconsistency in Fast Healthcare Interoperability Resources format utilitzation across institutions, despite requirements of the 21st Century Cures Act. Further testing across more institutions and annual exercises leveraging the application of data exchange over a blockchain infrastructure are recommended actions for determining the feasibility of this approach during a public health emergency and broaden the understanding of technical requirements for multisite data extraction.Conclusion:The FDA''s RAPID (Real-Time Application for Portable Interactive Devices) program, in collaboration with Discovery, the Critical Care Research Network''s PREP (Program for Resilience and Emergency Preparedness), identified the technical and legal challenges and requirements for rapid data exchange to a government entity using the FDA blockchain infrastructure.

In the previous few decades, the world has been challenged by a barrage of public health emergencies (PHEs), from natural disasters to the infectious disease threats of SARS (severe acute respiratory syndrome), H1N1, Zika, Ebola, and the COVID-19 pandemic. We have learned that PHEs are imminent and that the need for preparedness is paramount to our nation''s resiliency.1In the wake of COVID-19, widespread data collection to understand the virus''s impact and effectiveness of treatment plans are needed. However, the United States'' ability to rapidly collect multisite patient data to understand the impact of a disease and develop a unified and effective response remains a considerable vulnerability despite significant health system and federal investment in electronic health records (EHRs).2,3 The all-hazardscore data set, created in 2015 to characterize serious illness,injuries, and resource requirements to devise a robustresponse to PHEs, remains a challenge to collect giventechnological and regulatory limitations3 in regard to datasharing. This has been observed in the response to COVID-19, where the lack of data to create consensus on effective treatment protocols has been hindered.4–6Several barriers exist to data sharing in PHEs, including academic competition and inadequate human and technological resources during responses to emergency.7–10 Neither a standard approach to data sharing nor a method to negotiate and enforce the requisite data legal agreements exists.11,12 Moreover, effective methods for addressing deficiencies or advancing data sharing in response to PHEs are lacking.12–14 A clear need exists to explore novel methods to secure data collection to bridge the gap in knowledge sharing during PHEs.The complexity of data sharing from disparate sources is a problem experienced in other industries. The finance sector requires the highest level of security to manage financial transactions with speed and integrity. Blockchain technology emerged in the finance industry as a disruptive technology aimed at facilitating a decentralized, secure, and distributed ledger of transactions on a global scale.15,16 Blockchain technology works as blocks of information across a computer network; when chained together, these blocks create a single data asset.Blockchain has been suggested as an information infrastructure that can be used to advance knowledge sharing in the public sector.17 The decentralized nature of blockchain allows for interoperability,15 which is a key functionality needed to enable data sharing among hospital systems. The use of blockchain in medicine has the potential to revolutionize healthcare''s approach to data access, storage, and security17–19 by providing a method to share confidential patient information across many sites regardless of the local technical infrastructure. Large-scale data sharing would contribute to more robust medical research, advanced analytics (e.g., artificial intelligence), and the ability to benchmark the quality of care across institutions.The Food and Drug Administration (FDA) partnered with the Society of Critical Care Medicine''s Discovery, the Critical Care Research Network''s Program for Resilience and Emergency Preparedness (PREP; referred to as “Discovery PREP” hereafter) to explore the feasibility of using blockchain for multisite healthcare data collection in preparation for the required rapid data sharing during a PHE. Discovery PREP is one of many networks forming the Resilience Intelligence Network (RIN) with a combined focus on the nation''s resilience, preparedness, and response.2Discovery PREP and the FDA Real-Time Application for Portable Devices (RAPID) program20 collaborated to test the use of RAPID''s blockchain technology to determine the technical, legal, and resource challenges in the healthcare context. The RAPID program was designed to facilitate the automated extraction of key information from EHR systems needed to respond to adverse events without adding to the burden of data collection on healthcare practitioners.