摘要：Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

摘要：Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

摘要：Despite the availability of vaccines and antiviral therapies, seasonal influenza infections cause 400,000 human deaths on average per year. Low vaccine coverage and the occurrence of drug-resistant viral strains highlight the need for new and improved countermeasures. While influenza A virus (IAV) engineered to express a reporter gene may serve as a valuable tool for real-time tracking of viral infection, reporter gene insertion into IAV typically attenuates viral pathogenicity, hindering its application to research. Here, we demonstrate that lethal or even sublethal doses of bioluminescent IAV carrying the NanoLuc gene in the C-terminus of PB2 can be tracked in real-time in live mice without compromising pathogenicity. Real-time tracking of this bioluminescent IAV enables spatiotemporal viral replication tracking in animals that will facilitate the development of countermeasures by enhancing the interpretation of clinical signs and prognosis while also allowing less animal usage.

摘要：Objective. to determine the characterization of chondrogenic properties of adeno-associated virus type 2 (aaV2)- delivered hFgF18, via analysis of effects on primary human chondrocyte proliferation, gene expression, and in vivo cartilage thickness changes in the tibia and meniscus. Design. Chondrogenic properties of aaV2-FgF18 were compared with recombinant human FgF18 (rhFgF18) in vitro relative to phosphate-buffered saline (PBS) and aaV2-gFP negative controls. transcriptome analysis was performed using rNa-seq on primary human chondrocytes treated with rhFgF18 and aaV2- FgF18, relative to PBS. Durability of gene expression was assessed using aaV2-nluc and in vivo imaging. Chondrogenesis was evaluated by measuring weight-normalized thickness in the tibial plateau and the white zone of the anterior horn of the medial meniscus in Sprague-Dawley rats. Results. aaV2-FgF18 elicits chondrogenesis by promoting proliferation and upregulation of hyaline cartilage–associated genes, including COl2a1 and HaS2, while downregulating fibrocartilage- associated COl1a1. this activity translates to statistically significant, dose-dependent increases in cartilage thickness in vivo within the area of the tibial plateau, following a single intra-articular injection of the aaV2-FgF18 or a regimen of 6 twice-weekly injections of rhFgF18 protein relative to aaV2-gFP. in addition, we observed aaV2-FgF18-induced and rhFgF18-induced increases in cartilage thickness of the anterior horn of the medial meniscus. Finally, the single-injection aaV2-delivered hFgF18 offers a potential safety advantage over the multi-injection protein treatment as evidenced by reduced joint swelling over the study period. Conclusion. aaV2-delivered hFgF18 represents a promising strategy for the restoration of hyaline cartilage by promoting extracellular matrix production, chondrocyte proliferation, and increasing articular and meniscal cartilage thickness in vivo after a single intra-articular injection.

摘要：Ebola virus (EBOV) causes lethal disease in humans but not in mice. Here, we generated recombinant mouse-adapted (MA) EBOVs, including 1 based on the previously reported serially adapted strain (rMA-EBOV), along with single-reporter rMA-EBOVs expressing either fluorescent (ZsGreen1 [ZsG]) or bioluminescent (nano-luciferase [nLuc]) reporters, and dual-reporter rMA-EBOVs expressing both ZsG and nLuc. No detriment to viral growth in vitro was seen with inclusion of MA-associated mutations or reporter proteins. In CD-1 mice, infection with MA-EBOV, rMA-EBOV, and single-reporter rMA-EBOVs conferred 100% lethality; infection with dual-reporter rMA-EBOV resulted in 73% lethality. Bioluminescent signal from rMA-EBOV expressing nLuc was detected in vivo and ex vivo using the IVIS Spectrum CT. Fluorescent signal from rMA-EBOV expressing ZsG was detected in situ using handheld blue-light transillumination and ex vivo through epi-illumination with the IVIS Spectrum CT. These data support the use of reporter MA-EBOV for studies of Ebola virus in animal disease models.

摘要：Prior to the implementation of SARS CoV-2 mRNA based-vaccines, there was significant progress made in the research and development of mRNA vaccines against infectious diseases in animal models. These include influenza viruses, Zika virus, rabies virus, and HIV-1. In the past decade, researchers have made improvements to enhance protein translation, modulate immunogenicity, and improve delivery of mRNA therapies and vaccines. . Current methods for developing RNA vaccines use host cell machinery to translate mRNA-encoded target immunogens or therapeutic proteins. These methods also use lipid nanoparticle formulations to successfully deliver mRNAs encoding vaccine antigens or therapeutic proteins, and small interfering RNAs (siRNAs) to the cytosol.More recently, Pfizer, BioNTech and Moderna vaccines for SARS CoV-2 validated the potential of mRNA-lipid nanoparticle (LNP) technology. However, developing these vaccines also highlighted challenges with clinical-enabling manufacturing processes to produce these products. Current production methods are either larger scale and undisclosed, or bench-scale and unsuitable for early phase clinical studies. As it stands, there is a need for a scalable approach that is affordable and GMP compliant.This study sought to develop a platform for production that could be used at a lab scale and would fall under GMP conditions for the transition to early phase clinical studies. The methods they developed used non-replicating mRNA technology and incorporated a modified nucleoside. Researchers also optimized 5´ and 3´ UTR sequences, had a defined poly(A) tail length, integrated a cap analogue, and improved codon usage. These changes increased half-life, improved translation efficiency, and enhanced RNA stability and translation. Their scalable process was aqueous-based and easily adaptable to GMP manufacturing, and researchers have developed necessary analytical methods for stability testing, quality control and product characterization. Though the study highlights barriers to distribution, such as a need for long-duration stability, mRNA products have improved much in recent years, and results of this study show products developed with these methods provide good potency and protection in animal models.

摘要：Unidentified human remains (UHR) cases pose a global challenge for investigators and forensic analysts. In the US alone, over 14,000 UHR cases were open as of November 2022, according to the National Missing and Unidentified Persons System (NamUS). While newer technology, such as massively parallel sequencing, has enabled some improvements in identifying UHRs, short tandem repeat (STR) methods remain the most popular and widely used analytical techniques.The current study examined data from 121 UHR cases using manual DNA extraction: digestion with proteinase K/SDS, followed by extraction with organic solvents and DNA capture on silica spin columns. The extracted DNA was subjected to STR amplification using the PowerPlex^® 21 System. The researchers compared various sample types, including blood spots, long bones, skull bones and teeth, other bones, and tissues. Overall, the researchers reported an 89.2% success rate in identifying the UHRs using kinship analysis. Blood spot samples yielded the highest success rates with no failures among the samples tested. The researchers noted that, with skull/teeth samples, supplemental testing will likely be helpful in resolving identification challenges from the STR profile.

摘要：Previous studies have shown that individuals with and without cancer have different microRNA (miRNA) expression levels. MiRNAs are 20–25 nucleotide noncoding RNAs that regulate gene expression by inducing mRNA degradation and inhibiting mRNA transcription. Early cancer detection and treatment improves survival rates but screening for multiple cancers require multiple samples from patients. A blood-based screening method would reduce the screening burden on patients. Technologies like next-generation sequencing (NGS) make it easy to examine the expression profiles of over 2,500 miRNAs at once. However, previous miRNA cancer association studies produced inconsistent results that could be due to variations in blood handling and storage.This study examined the effects of anticoagulants and storage conditions on mRNA expression to standardize a protocol that could better provide reproducible NGS profiling data. Blood samples were gathered from healthy individuals to evaluate collection conditions and find the best parameters for reproducible miRNA results. Plasma samples were prepared and RNA extracted using automated methods before preparing cDNA libraries for miRNA expression analysis. Library concentration, identification rate, the unique molecular identifier (UMI) ratio and expression levels of erythrocyte- and platelet-derived miRNAs were evaluated. The researchers determined collecting blood in EDTA tubes had the highest RNA concentration and exhibited the lowest lysis compared to sodium fluoride and sodium citrate tubes. Storage time and temperature of both whole blood and plasma were tested to determine the best storage and handling conditions. The conditions tested were blood stored at room temperature from 1 hours to 3 days, plasma stored at 4°C and –20°C from 1 hour to 30 days and plasma banked at –80°C over 3, 4 and 5 years. During the study, several miRNAs were identified as time-dependent markers that increased or decreased over time in whole blood.Based on the results of blood and plasma samples, researchers applied what they learned to blood samples from cancer patients. Three facilities were asked to collect 20 blood samples using EDTA blood collection tubes, refrigerate within 1 hour, separate plasma within 12 hours, and store the plasma samples at –80°C. There were no significant differences in the 60 samples collected from the facilities based on library concentration, identification rate and UMI ratio. However, there was some variation in hemolysis among the facilities, but did not exceed the variation observed from samples drawn at the same facility. Researchers concluded that a reproducible miRNA expression profile was possible based on the blood handling and storage conditions they tested during their study.

摘要：Breast cancer (BC) is the most prevalent cancer worldwide, and mortality remains high in low-income countries due to few options for early detection. For individuals with dense breast tissue, the current method for early BC detection involves mammography plus ultrasound-assisted core needle biopsy for BC diagnosi s and staging. However, mammography has low sensitivity with a high number of false negatives for dense breast tissue and needle biopsies may miss small lesions. A noninvasive information-rich method that could more accurately diagnose cancer and optimize therapy could avoid these drawbacks. Prior studies of circulating tumor DNA (ctDNA), small nucleic acid fragments of 134–144bp, have shown high concordance between the ctDNA molecular profile and traditional tumor tissue testing methods. Next-generation sequencing (NGS) has simplified and improved the speed for identifying and testing ctDNA genomic alterations, which could improve patient outcomes.This study investigated what molecular markers might be associated with breast cancer in 32 patients that were sorted into four groups: mammography positive/needle biopsy negative diagnosis (pos-neg), positive for both mammography and needle biopsy (pos-pos) plus segmenting the positive breast cancer diagnosis into Class I (pos-pos I) and Class II (pos-pos II). Plasma samples from individuals were taken after the initial mammography and sorted into various groups after the needle biopsy results. Tumor-derived DNA levels was estimated before the microbiopsy but not found to be statically significant among the four groups after biopsy results, but there was a larger mean tumor DNA was present in the pos-pos stage II individuals.Next-generation sequencing and data analysis of ctDNA were used to determine which copy number alterations (CNA) single nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) were present and potentially associated with the diagnostic groupings. The results were further analyzed for cancer genes, coding and noncoding variants. The pos-neg individuals had deletion and gain CNAs that overlapped across 11 known oncogenes identified from other cancers plus three deletion CNAs of associated with three breast cancer oncogenes. For pos-pos individuals, five gain CNAs were found in five different breast-cancer associated oncogenes.The pos-pos group also had 10 SNP and indel variants that were associated with cancer, including one that is a known breast cancer risk factor located in SERAC1, which plays a role in splicing. Another variant occurred in F-box protein FBXW4. Decreased expression of FBXW4 correlated with poor prognosis for patients with other cancer types. Another pos-pos variant was identified in telomere function, one identified with a role in inflammation while the remaining variants were associated with known breast cancer risk genes. For the pos-neg group, there were six oncogene SNP and Indel variants identified that are associated with breast cancer, including a calcium-dependent protein kinase. While this study had limitations including a small sample size, no normal tissue testing from each subject and no healthy control individuals, researchers identified some CNAs, SNPs and Indels associated exclusively with either the pos-neg or pos-pos groups that look promising as a potential noninvasive early test for breast cancer.

摘要：The Gram-positive bacillus Clostridioides (formerly Clostridium) difficile is responsible for most hospital-related infections, with global incidence rising over the past decade. Two large toxins, toxin A (TcdA) and toxin B (TcdB), are responsible for triggering host responses that can result in significant intestinal damage. There is an urgent need for sensitive and rapid tests to detect true C. difficile infection, as distinguished from disease-free carriers.In this study, the researchers used NanoBiT^® technology to design “split-luciferase” assays that could detect TcdB and another C. difficile biomarker, glutamate dehydrogenase (GDH). The assay development strategy used affimers (synthetic nonimmunoglobulin-binding proteins) with high binding affinity for different regions of TcdB and GDH. These affimers are more convenient than antibodies, because they are smaller, stable and easily expressed as fusions with LgBiT and SmBiT tags. Binding to the target antigen in solution reconstitutes functional NanoBiT^® enzyme, resulting in a bright luminescent signal. The assay performance with fecal samples was equivalent to that of a current point-of-care (POC) test, but it had the advantages of being quantitative, requiring less user steps, and being able to distinguish clinically relevant TcdB. The researchers conclude that their assay method is suitable for a wide range of biomarkers and offers the potential to develop ultrasensitive and rapid POC tests for many infectious diseases.