National Strategic Research Institute

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan, China in late 2019, and its resulting coronavirus disease, COVID-19, was declared a pandemic by the World Health Organization on March 11, 2020. The rapid global spread of COVID-19 represents perhaps the most significant public health emergency in a century. As the pandemic progressed, a continued paucity of evidence on routes of SARS-CoV-2 transmission has resulted in shifting infection prevention and control guidelines between classically-defined airborne and droplet precautions. During the initial isolation of 13 individuals with COVID-19 at the University of Nebraska Medical Center, we collected air and surface samples to examine viral shedding from isolated individuals. We detected viral contamination among all samples, supporting the use of airborne isolation precautions when caring for COVID-19 patients.

Abstract Lack of evidence on SARS-CoV-2 transmission dynamics has led to shifting isolation guidelines between airborne and droplet isolation precautions. During the initial isolation of 13 individuals confirmed positive with COVID-19 infection, air and surface samples were collected in eleven isolation rooms to examine viral shedding from isolated individuals. While all individuals were confirmed positive for SARS-CoV-2, symptoms and viral shedding to the environment varied considerably. Many commonly used items, toilet facilities, and air samples had evidence of viral contamination, indicating that SARS-CoV-2 is shed to the environment as expired particles, during toileting, and through contact with fomites. Disease spread through both direct (droplet and person-to-person) as well as indirect contact (contaminated objects and airborne transmission) are indicated, supporting the use of airborne isolation precautions. One Sentence Summary SARS-CoV-2 is shed during respiration, toileting, and fomite contact, indicating that infection may occur in both direct and indirect contact.

BACKGROUND: Aerosol transmission of COVID-19 is the subject of ongoing policy debate. Characterizing aerosol produced by people with COVID-19 is critical to understanding the role of aerosols in transmission. OBJECTIVE: We investigated the presence of virus in size-fractioned aerosols from six COVID-19 patients admitted into mixed acuity wards in April of 2020. METHODS: Size-fractionated aerosol samples and aerosol size distributions were collected from COVID-19 positive patients. Aerosol samples were analyzed for viral RNA, positive samples were cultured in Vero E6 cells. Serial RT-PCR of cells indicated samples where viral replication was likely occurring. Viral presence was also investigated by western blot and transmission electron microscopy (TEM). RESULTS: SARS-CoV-2 RNA was detected by rRT-PCR in all samples. Three samples confidently indicated the presence of viral replication, all of which were from collected sub-micron aerosol. Western blot indicated the presence of viral proteins in all but one of these samples, and intact virions were observed by TEM in one sample. SIGNIFICANCE: Observations of viral replication in the culture of submicron aerosol samples provides additional evidence that airborne transmission of COVID-19 is possible. These results support the use of efficient respiratory protection in both healthcare and by the public to limit transmission.

Liquid metal-elastomer composite is a promising soft conductor for skin-interfaced bioelectronics, soft robots, and others due to its large stretchability, ultrasoftness, high electrical conductivity, and mechanical-electrical decoupling. However, it often suffers from deformation-induced leakage, which can smear skin, deteriorate device performance, and cause circuit shorting. Besides, antimicrobial property is desirable in soft conductors to minimize microbial infections. Here, we report phase separation-based synthesis of porous liquid metal-elastomer composites with high leakage resistance and antimicrobial property, together with large stretchability, tissue-like compliance, high and stable electrical conductivity over deformation, high breathability, and magnetic resonance imaging compatibility. The porous structures can minimize leakage through damping effects and lower percolation thresholds to reduce liquid metal usage. In addition, epsilon polylysine is loaded into elastic matrices during phase separation to provide antimicrobial property. The enabled skin-interfaced bioelectronics can monitor cardiac electrical and mechanical activities and offer electrical stimulations in a mechanically imperceptible and electrically stable manner even during motions.

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission causing coronavirus disease 2019 (COVID-19) may occur through multiple routes. We collected aerosol samples around six patients admitted into mixed acuity wards in April of 2020 to identify the risk of airborne SARS-CoV-2. Measurements were made to characterize the size distribution of aerosol particles, and size-fractionated, aerosol samples were collected to assess the presence of infectious virus in particles sizes of >4.1 µm, 1-4 µm, and <1 µm in the patient environment. Samples were analyzed by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR), cell culture, western blot, and transmission electron microscopy (TEM). SARS-CoV-2 RNA was detected in all six rooms in all particle size fractions (>4.1 µm, 1-4 µm, and <1 µm). Increases in viral RNA during cell culture of the virus from recovered aerosol samples demonstrated the presence of infectious, replicating virions in three <1 µm aerosol samples (P<0.05). Viral replication of aerosol was also observed in the 1-4 µm stage but did not reach statistical significance (0.05<P<0.10). Western blot and TEM analysis of these samples also showed evidence of viral proteins and intact virions. The infectious nature of aerosol collected in this study further suggests that airborne transmission of COVID-19 is possible, and that aerosol prevention measures are necessary to effectively stem the spread of SARS-CoV-2.

The highly infectious and zoonotic pathogen Francisella tularensis is the etiologic agent of tularemia, a potentially fatal disease if untreated. Despite the high average nucleotide identity, which is >99.2% for the virulent subspecies and >98% for all four subspecies, including the opportunistic microbe Francisella tularensis subsp. novicida , there are considerable differences in genetic organization. These chromosomal disparities contribute to the substantial differences in virulence observed between the various F. tularensis subspecies and subtypes.

Over the past few decades, it has been well established that gut microbiota-derived metabolites can disrupt gut function, thus resulting in an array of diseases. Notably, phenylacetylglutamine (PAGln), a bacterial derived metabolite, has recently gained attention due to its role in the initiation and progression of cardiovascular and cerebrovascular diseases. This meta-organismal metabolite PAGln is a byproduct of amino acid acetylation of its precursor phenylacetic acid (PAA) from a range of dietary sources like egg, meat, dairy products, etc. The microbiota-dependent metabolism of phenylalanine produces PAA, which is a crucial intermediate that is catalyzed by diverse microbial catalytic pathways. PAA conjugates with glutamine and glycine in the liver and kidney to predominantly form phenylacetylglutamine in humans and phenylacetylglycine in rodents. PAGln is associated with thrombosis as it enhances platelet activation mediated through the GPCRs receptors α2A, α2B, and β2 ADRs, thereby aggravating the pathological conditions. Clinical evidence suggests that elevated levels of PAGln are associated with pathology of cardiovascular, cerebrovascular, and neurological diseases. This Review further consolidates the microbial/biochemical synthesis of PAGln and discusses its role in the above pathophysiologies.

Cyber-Physical Systems (CPS) represent a fundamental link between information technology (IT) systems and the devices that control industrial production and maintain critical infrastructure services that support our modern world. Increasingly, the interconnections among CPS and IT systems have created exploitable security vulnerabilities due to a number of factors, including a legacy of weak information security applications on CPS and the tendency of CPS operators to prioritize operational availability at the expense of integrity and confidentiality. As a result, CPS are subject to a number of threats from cyber attackers and cyber-physical attackers, including denial of service and even attacks against the integrity of the data in the system. The effects of these attacks extend beyond mere loss of data or the inability to access information system services. Attacks against CPS can cause physical damage in the real world. This paper reviews the challenges of providing information assurance services for CPS that operate critical infrastructure systems and industrial control systems. These methods are thorough measures to close integrity and confidentiality gaps in CPS and processes to highlight the security risks that remain. This paper also outlines approaches to reduce the overhead and complexity for security methods, as well as examine novel approaches, including covert communications channels, to increase CPS security.

Following the COVID-19 outbreak, swabs for biological specimen collection were thrust to the forefront of healthcare materials. Swab sample collection and recovery are vital for reducing false negative diagnostic tests, early detection of pathogens, and harvesting DNA from limited biological samples. In this study, we report a new class of nanofiber swabs tipped with hierarchical 3D nanofiber objects produced by expanding electrospun membranes with a solids-of-revolution-inspired gas foaming technique. Nanofiber swabs significantly improve absorption and release of proteins, cells, bacteria, DNA, and viruses from solutions and surfaces. Implementation of nanofiber swabs in SARS-CoV-2 detection reduces the false negative rates at two viral concentrations and identifies SARS-CoV-2 at a 10× lower viral concentration compared to flocked and cotton swabs. The nanofiber swabs show great promise in improving test sensitivity, potentially leading to timely and accurate diagnosis of many diseases.

A vaccine for smallpox is no longer administered to the general public, and there is no proven, safe treatment specific to poxvirus infections, leaving people susceptible to infections by smallpox and other zoonotic Orthopoxviruses such as monkeypox. Using vaccinia virus (VACV) as a model organism for other Orthopoxviruses, CRISPR-Cas9 technology was used to target three essential genes that are conserved across the genus, including A17L, E3L, and I2L. Three individual single guide RNAs (sgRNAs) were designed per gene to facilitate redundancy in rendering the genes inactive, thereby reducing the reproduction of the virus. The efficacy of the CRISPR targets was tested by transfecting human embryonic kidney (HEK293) cells with plasmids encoding both SaCas9 and an individual sgRNA. This resulted in a reduction of VACV titer by up to 93.19% per target. Following the verification of CRISPR targets, safe and targeted delivery of the VACV CRISPR antivirals was tested using adeno-associated virus (AAV) as a packaging vector for both SaCas9 and sgRNA. Similarly, AAV delivery of the CRISPR antivirals resulted in a reduction of viral titer by up to 92.97% for an individual target. Overall, we have identified highly specific CRISPR targets that significantly reduce VACV titer as well as an appropriate vector for delivering these CRISPR antiviral components to host cells in vitro.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CPS-enabled systems form the underlying support infrastructure that impact the efficient delivery of critical goods and services in our modern society. Failure to adequately protect the integrity, confidentiality, and availability of our critical infrastructure systems, like transportation, delivery of energy and water, and healthcare, could introduce vulnerabilities that could be exploited by attackers. CPS networks originated from a number of proprietary protocols, built to operate without connections that extended beyond the plant walls. This “security through obscurity” approach did not reveal widespread security risks until these systems were interconnected with other information technology systems, including vulnerable business operations networks. The characteristics of the communications protocols used in operational technology systems, like Modbus, present a number of barriers to adopting security enhancements. When these systems are exploited, which can be easily demonstrated on a Modbus system, the impact of security failures can be severe. Covert channels have the potential to create an out-of-band communication path that would enable some level of integrity checking between devices. The Modbus Covert Channel Integrity Check is a proposed concept that would demonstrate the use of covert channels as a method of secure communication that would prevent a number of attacks, including man-in-the-middle.

The study of infectious diseases includes both the progression of the disease in its host and how it transmits between hosts. Understanding disease transmission is important for recommending effective interventions, protecting healthcare workers, and informing an effective public health response. Sampling the environment for infectious diseases is critical to public health since it can provide an understanding of the mechanisms of transmission, characterization of contamination in hospitals and other public areas, and the spread of a disease within a community. Measurements of biological aerosols, particularly those that may cause disease, have been an ongoing topic of research for decades, and so a wide variety of technological solutions exist. This wide field of possibilities can create confusion, particularly when different approaches yield different answers. Therefore, guidelines for best practice in this area are important to allow more effective use of this data in public health decisions. This review examines air, surface and water/wastewater sampling methods, with a focus on aerosol sampling, and a goal of recommending approaches to designing and implementing sampling systems that may incorporate multiple strategies. This is accomplished by developing a framework for designing and evaluating a sampling strategy, reviewing current practices and emerging technologies for sampling and analysis, and recommending guidelines for best practice in the area of aerosol sampling for infectious disease.

Circumstances exist that call for the aeromedical evacuation high-level containment transport (AE-HLCT) of patients with highly hazardous communicable diseases. A small number of organizations maintain AE-HLCT capabilities, and little is publicly available regarding the practices. The time is ripe for the development of standards and consensus guidelines involving AE-HLCT.

Remote, terrestrial, and space sensors require sources that have high enough power and energy densities for continuous operation for multiple decades. Conventional chemical sources have lower energy densities and lifetimes of 10 to 15 years depending on environmental conditions. Betavoltaic (βV) nuclear batteries using β--emitting radioisotopes possess energy densities approximately 1000 times greater than conventional chemical sources. Their electrical power density (Pe,vol in W/cm3) in a given volume is a function of β--flux surface power density , surface interface type between radioisotope and transducer, β- range, and transducer thickness and conversion efficiency (ηs). Tritium is the most viable β--emitting radioisotope because of its commercial availability, low biotoxicity, half-life, and low energy, which minimizes the penetration depth and damage of transducer. To maximize Pe,vol, tritium in solid or liquid form must be used in the βV nuclear battery. A Monte Carlo source model using MCNP6 was developed to maximize the Pe,vol of a tritium-based βV nuclear battery. First, a planar coupling configuration with different tritiated compounds (ie, titanium tritide and tritiated nitroxide) and a semiconductor transducer (4H-SiC) with thicknesses of 1 and 100 μm were modeled. The results showed that β--source efficiency (ηβ), which is the percentage of energy deposited in the transducer, decreased as the tritiated compound's mass density increased. The highest Pe,vol was dependent on a combination of characteristics: specific activity (Am in Ci/g), mass density, and 4H-SiC layer thickness. The tritiated nitroxide with the highest Am at 2372 Ci/g produced the highest Pe,vol at 2.46 mW/cm3. Second, a 3-D coupling configuration was modelled to increase surface interfacing between the radioisotope source and textured transducer surface. 3-D coupling configuration increased the percentage of energy deposited into the transducer because of more surface interfacing between the transducer and source in the same volume. The tritiated nitroxide was selected as the radioisotope source coupled with five different textured surface feature types. The Pe,vol as a function of textured surface feature and gap, where the radioisotope is located, width was calculated for 1- and 100-μm 4H-SiC layer thicknesses. Results showed that ηβ increased compared with planar coupling configuration (ie, approximately 56.2% increase over planar with cylindrical hole array) except with the rectangular pillar array. Still, the rectangular pillar array produced the highest Pe,vol at 4.54 mW/cm3 with an increasing factor of 2.29 compared with the planar coupling configuration.

DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by subtle mechanistic differences and limited understanding of DNA replication in pathogenic microorganisms. Clostridium difficile is the main cause of healthcare-associated diarrhoea and its DNA replication machinery is virtually uncharacterized. We identify and characterize the mechanistic details of the putative replicative helicase (CD3657), helicase-loader ATPase (CD3654) and primase (CD1454) of C. difficile, and reconstitute helicase and primase activities in vitro We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we find that helicase activity is dependent on the presence of primase in vitro The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader-helicase-primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model.

, can be used to detect the helical structures of DNA molecules in biological systems, however, no CIDS measurement from single particles has been reported to date. We report an innovative method for measuring CIDS phase functions from single particles individually flowing through a scattering laser beam. CIDS signals were obtained from polystyrene latex (PSL) microspheres with or without coating of DNA molecules, tryptophan particles, and aggregates of B. subtilis spores, at the size of 3 μm in diameter. Preliminary results show that this method is able to measure CIDS phase function in tens of microseconds from single particles, and has the ability to identify particles containing biological molecules.

Abstract As the world recovers from the lockdown imposed by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, returning to shared indoor spaces is considered a formidable risk. It is now clear that transmission of SARS-CoV-2 is driven by respiratory microdroplets expelled by infected persons, which can become suspended in the air. Several layering technologies are being explored to mitigate indoor transmission in the hopes of re-opening business, schools and transportation systems. Here we coupled the water adsorptive and photocatalytic capacity of novel Metal Organic Frameworks (MOFs) to demonstrate the capture and inactivation of SARS-CoV-2. Discussion is given on the methods of analysis and the differences between the photocatalytic activity of several MOFs, and the difference between MOF induced photocatalysis and ultra violet photolysis of SARS-CoV-2. Our results are intended to provide support to industry looking for alternative methods secure indoor spaces.

ABSTRACTWe present an advanced optical method to measure the phase function of circular intensity differential scattering (CIDS), i.e., the normalized Mueller matrix element -S14/S11, from individual single flowing through aerosol particles. Here, a 32-anode photomultiplier tube and its associated electronics, combined with an elliptical reflector, were used to record the scattering phase functions, when a particle were illuminated by a left-handed and a right-handed circular polarization laser beam around the focus of the reflector successively. The new design does not need lock-in amplifier, polarization modulator, and rotating goniometer as the traditional setup. It can reach a particle detection ability with a maximum rate of 50,000 particle/sec. CIDS phase functions from tryptophan particles, polystyrene latex microspheres, aggregates of Escherichia coli, Bacillus subtilis spores, Yersinia rohdei, and bacteriophage MS2 were measured, the results showed that this method has the ability to rapidly discriminate between single bioaerosol and non-bioaerosol particles.Keywords: Circular intensity differential scattering (CIDS)Mueller matrix element -S14/S11 Bioaerosol particlesChiralitySpiralElastic light scatteringScattering phase functionDisclaimerAs a service to authors and researchers we are providing this version of an accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proofs will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to these versions also.

Maternal opioid use poses a significant health concern not just to the expectant mother but also to the fetus. Notably, increasing numbers of children born suffering from neonatal opioid withdrawal syndrome (NOWS) further compounds the crisis. While epidemiological research has shown the heightened risk factors associated with NOWS, little research has investigated what molecular mechanisms underly the vulnerabilities these children carry throughout development and into later life. To understand the implications of in utero and post-natal opioid exposure on the developing brain, we sought to assess the response to one of the most common pediatric injuries: minor traumatic brain injury (mTBI). Using a rat model of in utero and post-natal oxycodone (IUO) exposure and a low force weight drop model of mTBI, we show that not only neonatal opioid exposure significantly affects neuroinflammation, brain metabolites, synaptic proteome, mitochondrial function, and altered behavior in juvenile rats, but also, in conjunction with mTBI these aberrations are further exacerbated. Specifically, we observed long term metabolic dysregulation, neuroinflammation, alterations in synaptic mitochondria, and impaired behavior were impacted severely by mTBI. Our research highlights the specific vulnerability caused by IUO exposure to a secondary stressor such as later life brain injury. In summary, we present a comprehensive study to highlight the damaging effects of prenatal opioid abuse in conjunction with mild brain injury on the developing brain.