Evaluation of Commercial RNA Extraction Protocols for Avian Influenza Virus Using Nanopore Metagenomic Sequencing.

Avian influenza virus (AIV) is a significant threat to the poultry industry, necessitating rapid and accurate diagnosis. The current AIV diagnostic process relies on virus identification via real-time reverse transcription-polymerase chain reaction (rRT-PCR). Subsequently, the virus is further characterized using genome sequencing. This two-step diagnostic process takes days to weeks, but it can be expedited by using novel sequencing technologies. We aim to optimize and validate nucleic acid extraction as the first step to establishing Oxford Nanopore Technologies (ONT) as a rapid diagnostic tool for identifying and characterizing AIV from clinical samples. This study compared four commercially available RNA extraction protocols using AIV-known-positive clinical samples. The extracted RNA was evaluated using total RNA concentration, viral copies as measured by rRT-PCR, and purity as measured by a 260/280 absorbance ratio. After NGS testing, the number of total and influenza-specific reads and quality scores of the generated sequences were assessed. The results showed that no protocol outperformed the others on all parameters measured; however, the magnetic particle-based method was the most consistent regarding CT value, purity, total yield, and AIV reads, and it was less error-prone. This study highlights how different RNA extraction protocols influence ONT sequencing performance.

Disinformation and Regime Survival.

Disinformation has transformed into a global issue and while it is seen as a growing concern to democracy today, autocrats have long used it as a part of their propaganda repertoire. Yet, no study has tested the effect of disinformation on regime stability and breakdown beyond country-specific studies. Drawing on novel measures from the Digital Society Project (DSP) estimating the levels of disinformation disseminated by governments across 148 countries between 2000-2022 and from the Episodes of Regime Transformation (ERT) dataset, we provide the first global comparative study of disinformation and survival of democratic and authoritarian regimes, respectively. The results show that in authoritarian regimes, disinformation helps rulers to stay in power as regimes with higher levels of disinformation are less likely to experience democratization episodes. In democracies, on the other hand, disinformation increases the probability of autocratization onsets. As such, this study is the first to provide comparative evidence on the negative effects of disinformation on democracy as well as on the prospects of democratization.

An outbreak of SARS-CoV-2 omicron variant and deaths of three lions in a zoo.

There have been reports of the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to various mammalian species. Some infected animals show clinical signs and may even die in rare cases. Outbreaks of SARS-CoV-2 have been reported in zoos where susceptible animals are bred in high population densities. However, there have been few reports of omicron variant outbreaks in zoo animals. From late 2022 to 2023, an outbreak of the SARS-CoV-2 omicron variant occurred in one Japanese zoo. A total of 24 lions were housed in the zoo; 13 of them showed respiratory symptoms, and the three oldest lions died. Molecular and histopathological analyses revealed that the deceased lions were infected with SARS-CoV-2 omicron BF.7.15. Virus-neutralization tests showed that all 21 lions were positive for antibodies against the omicron variant, but not against the delta variant. In addition, three tigers and one bear in the same or neighboring building as the lions possessed antibodies against the omicron variant. This is a very rare report on the outbreak of a SARS-CoV-2 omicron variant infection that resulted in the death of animals. This finding demonstrates the importance of continuous countermeasures to protect non-vaccinated animals from SARS-CoV-2 infection.

Obesity-associated Inflammation and Alloimmunity.

Obesity is a worldwide health problem with a rapidly rising incidence. In organ transplantation, increasing numbers of patients with obesity accumulate on waiting lists and undergo surgery. Obesity is in general conceptualized as a chronic inflammatory disease, potentially impacting alloimmune response and graft function. Here, we summarize our current understanding of cellular and molecular mechanisms that control obesity-associated adipose tissue inflammation and provide insights into mechanisms affecting transplant outcomes, emphasizing on the beneficial effects of weight loss on alloimmune responses.

Development and Evaluation of Mycoplasma gallisepticum Challenge Model in Layer Pullets.

Manufacturers of Mycoplasma gallisepticum (MG) modified live vaccines usually recommend a single application at 8 wk of age. This makes 12-16-wk-old layer pullets suitable for challenge studies intended to evaluate these vaccines. Numerous challenge models in different poultry species and ages have been reported. However, there is not an established layer pullet challenge model for this age. The aim of this study is to develop a suitable challenge model in 12-wk-old layer pullets. MG Rlow strain was used as the challenge strain, and its ability to induce clinical signs and lesions in 12-wk-old Hy-Line W-36 layer pullets was evaluated. Three different doses (low, 7.95 × 104 color-changing units [CCU]/bird; medium, 7.95 × 106 CCU/bird; and high, 7.95 × 108 CCU/bird) via three different routes (eye drop, fine spray, and contact infection) were compared and evaluated using different parameters. At 14 days post-challenge, there were no mortalities in any of the groups throughout the study. Layer pullets directly challenged with the high dose via the fine spray route showed the clearest and most consistent results (clinical signs, positive quantitative real-time PCR [qPCR], seroconversion, air sac scoring, and histopathological changes of the tracheal mucosa). Medium and low challenge doses applied via fine spray or eye drop did not show consistent results. Rlow strain was able to spread to the contact infection birds, as confirmed by the positive qPCR results; however, none of the contact-infected birds showed any clinical signs or gross or microscopic lesions. Our results suggest that a high dose (7.95 × 108 CCU/bird) administered through a fine spray route is the model of choice in any future MG vaccine evaluation trials in 12-wk-old layer pullets.

Nota de investigación- Desarrollo y evaluación del modelo de desafío para Mycoplasma gallisepticum en pollitas de postura. Los fabricantes de vacunas vivas modificadas contra Mycoplasma gallisepticum (MG) suelen recomendar una sola aplicación a las ocho semanas de edad. Esto hace que las pollitas de postura de 12 a 16 semanas de edad sean adecuadas para estudios de desafío destinados a evaluar estas vacunas. Se han reportado numerosos modelos de desafío en diferentes especies y edades de aves de corral. Sin embargo, no existe un modelo de desafío establecido para pollitas de postura de esta edad. El objetivo de este estudio fue desarrollar un modelo de desafío adecuado en pollitas ponedoras de 12 semanas de edad. Se utilizó la cepa Rlow de Mycoplasma gallisepticum como cepa de desafío y se evaluó su capacidad para inducir signos clínicos y lesiones en pollitas ponedoras Hy-Line W-36 de 12 semanas de edad. Tres dosis diferentes (baja, 7.95 × 104 unidades de cambio de color [CCU]/ave; media, 7.95 × 106 CCU/ave; y alta, 7.95 × 108 CCU/ave) a través de tres rutas diferentes (gota en el ojo, aerosol con gota fina e infección por contacto) se compararon y evaluaron utilizando diferentes parámetros. A los 14 días posteriores al desafío, no hubo mortalidades en ninguno de los grupos durante todo el estudio. Las pollitas de postura expuestas directamente a la dosis alta a través de la ruta de aerosol con gota fina mostraron los resultados más claros y consistentes (signos clínicos, PCR cuantitativa en tiempo real [qPCR] positiva, seroconversión, puntuación de lesiones en los sacos aéreos y cambios histopatológicos de la mucosa traqueal). Las dosis de desafío medias y bajas aplicadas mediante aerosol con gota fina o gota en el ojo no mostraron resultados consistentes. La cepa Rlow pudo propagarse a las aves infectadas por contacto, como lo confirmaron los resultados positivos de qPCR; sin embargo, ninguna de las aves infectadas por contacto mostró signos clínicos o lesiones macroscópicas o microscópicas. Estos resultados sugieren que una dosis alta (7.95 × 108 CCU/ave) administrada a través de una ruta de aerosol con gota fina es el modelo de elección en cualquier ensayo futuro de evaluación de vacunas para M. gallisepticum en pollitas de postura de 12 semanas de edad.

Visualizing histone H4K20me1 in knock-in mice expressing the mCherry-tagged modification-specific intracellular antibody.

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into the Rosa26 locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types.

ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin.

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.

Mode of SUV420H2 heterochromatin localization through multiple HP1 binding motifs in the heterochromatic targeting module.

Constitutive heterochromatin is transcriptionally repressed and densely packed chromatin, typically harboring histone H3 Lys9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1). SUV420H2, a histone H4 Lys20 methyltransferase, is recruited to heterochromatin by binding to HP1 through its Heterochromatic Targeting Module (HTM). Here, we have identified three HP1 binding motifs within the HTM. Both the full-length HTM and its N-terminal region (HTM-N), which contains the first and second motifs, stabilized HP1 on heterochromatin. The intervening region between the first and second HP1 binding motifs in HTM-N was also crucial for HP1 binding. In contrast, the C-terminal region of HTM (HTM-C), containing the third motif, destabilized HP1 on chromatin. An HTM V374D mutant, featuring a Val374 to Asp substitution in the second HP1 binding motif, localizes to heterochromatin without affecting HP1 stability. These data suggest that the second HP1 binding motif in the SUV420H2 HTM is critical for locking HP1 on H3K9me3-enriched heterochromatin. HTM V374D, tagged with a fluorescent protein, can serve as a live-cell probe to visualize HP1-bound heterochromatin.

Impact of renal dysfunction on the diagnosis of acute pancreatitis using urinary trypsinogen-2: A retrospective study.

AIM: Early diagnosis of acute pancreatitis is crucial, and urinary trypsinogen has been recently reported as a useful biomarker for diagnosing acute pancreatitis. We aimed to evaluate the impact of renal dysfunction on the diagnostic performance of urinary trypsinogen-2 for acute pancreatitis. METHODS: We conducted a retrospective study using the clinical data of patients who visited the Department of Emergency and Critical Care at the University of Tokyo Hospital between 1 October, 2021, and 30 June, 2022. Patients with available data on qualitative urinary trypsinogen-2 levels were identified. We compared the urinary trypsinogen-2 levels among patients who were clinically diagnosed with acute pancreatitis. We further stratified the patients according to renal function parameters, such as serum creatinine level, blood urea nitrogen level, and estimated glomerular filtration rate, and evaluated the performance of urinary trypsinogen-2 as a biomarker for acute pancreatitis. RESULTS: Within 9 months, 35 patients were identified. Of them, 22 patients showed positive results and 13 showed negative results on the urinary trypsinogen-2 test. The sensitivity, specificity, positive predictive value, and negative predictive value were 0.80, 0.40, 0.18, and 0.92, respectively. Based on the blood urea nitrogen level and estimated glomerular filtration rate, the prevalence of false-positive results was significantly higher in patients with reduced renal function than in those with normal renal function. CONCLUSION: In patients with reduced renal function, the urinary trypsinogen-2 qualitative test results might be interpreted with caution when used for diagnosing acute pancreatitis.

Independent mesenchymal progenitor pools respectively produce and maintain osteogenic and chondrogenic cells in zebrafish.

Skeletal tissues including cartilage and bones are characteristic features of vertebrates that are crucial for supporting body morphology and locomotion. Studies mainly in mice have shown that osteoblasts and chondroblasts are supplied from several progenitors like the sclerotome cells in the embryonic stage, osteo-chondroprogenitors in growing long bones, and skeletal stem cells of bone marrow in the postnatal period. However, the exact origins of progenitor cells, their lineage relationships, and their potential to differentiate into osteoblasts and chondroblasts from embryos to adult tissues are not well understood. In this study, we conducted clonal cell tracking in zebrafish and showed that sox9a+ cells are already committed to either chondrogenic or osteogenic fates during embryonic stages and that respective progenies are independently maintained as mesenchymal progenitor pools. Once committed, they never change their lineage identities throughout animal life, even through regeneration. In addition, we further revealed that only osteogenic mesenchymal cells replenish the osteoblast progenitor cells (OPCs), a population of reserved tissue stem cells found to be involved in the de novo production of osteoblasts during regeneration and homeostasis in zebrafish. Thus, our clonal cell tracking study in zebrafish firstly revealed that the mesenchymal progenitor cells that are fated to develop into either chondroblasts or osteoblasts serve as respective tissue stem cells to maintain skeletal tissue homeostasis. Such mesenchymal progenitors dedicated to producing either chondroblasts or osteoblasts would be important targets for skeletal tissue regeneration.

Macacine alphaherpesvirus 1 (B Virus) Infection in Humans, Japan, 2019.

Two human patients with Macacine alphaherpesvirus 1 infection were identified in Japan in 2019. Both patients had worked at the same company, which had a macaque facility. The rhesus-genotype B virus genome was detected in cerebrospinal fluid samples from both patients.

Utility of Feathers for Avian Influenza Virus Detection in Commercial Poultry.

The present study evaluated the potential utility of feather samples for the convenient and accurate detection of avian influenza virus (AIV) in commercial poultry. Feather samples were obtained from AIV-negative commercial layer facilities in Iowa, USA. The feathers were spiked with various concentrations (106 to 100) of a low pathogenic strain of H5N2 AIV using a nebulizing device and were evaluated for the detection of viral RNA using a real-time RT-PCR assay immediately or after incubation at -20, 4, 22, or 37 °C for 24, 48, or 72 h. Likewise, cell culture medium samples with and without the virus were prepared and used for comparison. In the spiked feathers, the PCR reliably (i.e., 100% probability of detection) detected AIV RNA in eluates from samples sprayed with 103 EID50/mL or more of the virus. Based on half-life estimates, the feathers performed better than the corresponding media samples (p < 0.05), particularly when the samples were stored at 22 or 37 °C. In conclusion, feather samples can be routinely collected from a poultry barn as a non-invasive alternative to blood or oropharyngeal-cloacal swab samples for monitoring AIV.

Development and psychometric testing of the Professional Interpersonal Competency Assessment Scale for Novice nurses (PICASN).

Background: Assessing the professional interpersonal competency of novice nurses is crucial for preventing staff turnover and promoting effective work. However, none of the instruments identified in the literature specifically target novice nurses. Objective: This study aimed to develop and psychometrically test the perception dimension of the Professional Interpersonal Competency Assessment Scale for Novice nurses (PICASN) in Japan. Methods: The study comprised four steps: 1) concept identification, 2) item construction, 3) validity measure, and 4) reliability measure. A cross-sectional web-based questionnaire was administered from February to April 2023 and was completed by 203 novice nurses. Data quality was assessed using mean, item response, missing values, floor and ceiling effects, internal consistency, and item-rest correlations. Content validity index (CVI) was used to determine the instrument's validity, while exploratory factor analysis (EFA) using maximum likelihood estimation with Promax rotation was employed to assess the factor structure. Cronbach's alpha was used to evaluate reliability. Results: The 27-item PICASN demonstrated an Item-CVI of 0.94 and a Scale-CVI of 0.88. EFA revealed two factors: 1) Basic competencies as a novice nurse (15 items) and 2) Relationship building skills within the healthcare team (12 items), which explained 80% of the variance. Internal consistency reliability was excellent at 0.94 and 0.91 for the factors, and the overall scale reliability was 0.95. The item-rest (I-R) correlation values exceeding 0.6 were considered acceptable. Conclusion: The PICASN demonstrates satisfactory psychometric properties, making it an effective tool for measuring professional interpersonal competency among novice nurses in Japan. This instrument serves to assist novice nurses by promoting self-awareness and offering targeted insights into specific areas requiring improvement. Additionally, it provides experienced nurses and nurse managers with valuable insights into team dynamics, guiding interventions for continuous quality improvement.

Differences between left adrenal vein sampling sites revealed with segmental sampling in primary aldosteronism.

OBJECTIVES: To clarify the differences between two left sampling sites (left adrenal central vein [LCV] and common trunk [CMT], conjunction with LCV and inferior phrenic vein) and their impact on adrenal venous sampling (AVS) in primary aldosteronism by analyzing the results of segmental AVS (sAVS). METHODS: We retrospectively analyzed a final cohort of 432 patients who underwent cosyntropin-stimulated sAVS from 2017 to 2020. Hormone levels in the LCV and the CMT were compared. Subtype diagnosis was based on the lateralization index with LCV and CMT sampling (a cutoff value of 4) and sAVS after excluding patients with a selectivity index (SI) <3. RESULTS: Compared with the LCV, CMT sampling showed significantly lower aldosterone and cortisol levels and a higher proportion of cases with an SI of <3 (2.8% vs 0.5%, p = 0.025) and <5 (6.9% vs 0.5%, p < 0.001), while the aldosterone-to-cortisol ratio and the lateralization index were not significantly different. Subtyping of both sites was concordant in 94.7% (393/415) and discordant in the remainder, which included left and right aldosterone-producing adenoma and idiopathic hyperaldosteronism cases referring to the sAVS. There was no significant difference between the concordance rate of the two sampling sites based on the sAVS diagnosis. CONCLUSIONS: LCV sampling meets the SI criteria for successful AVS more frequently compared with CMT sampling, but neither was better than the other in terms of diagnosis under conditions of meeting the criteria. ADVANCES IN KNOWLEDGE: LCV sampling would decrease the number of cases judged as AVS failure.

Complete genome sequences generated using hybrid Nanopore-Illumina assembly of two non-typical Avibacterium paragallinarum strains isolated from clinically normal chicken flocks.

We report the complete genome sequences of two non-typical Avibacterium paragallinarum (AP) strains isolated from chickens in the absence of clinical signs. The availability of these genomes can aid scientists in improving current diagnostics and increase our understanding of AP epidemiology and pathogenicity in chickens.

Recombinant spike protein vaccines coupled with adjuvants that have different modes of action induce protective immunity against SARS-CoV-2.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a glycoprotein, expressed on the virion surface, that mediates infection of host cells by directly interacting with host receptors. As such, it is a reasonable target to neutralize the infectivity of the virus. Here we found that a recombinant S protein vaccine adjuvanted with Alhydrogel or the QS-21-like adjuvant Quil-A effectively induced anti-S receptor binding domain (RBD) serum IgG and neutralizing antibody titers in the Syrian hamster model, resulting in significantly low SARS-CoV-2 replication in respiratory organs and reduced body weight loss upon virus challenge. Severe lung inflammation upon virus challenge was also strongly suppressed by vaccination. We also found that the S protein vaccine adjuvanted with Alhydrogel, Quil-A, or an AS03-like adjuvant elicited significantly higher neutralizing antibody titers in mice than did unadjuvanted vaccine. Although the neutralizing antibody titers against the variant viruses B.1.351 and B.1.617.2 declined markedly in mice immunized with wild-type S protein, the binding antibody levels against the variant S proteins were equivalent to those against wild-type S. When splenocytes from the immunized mice were re-stimulated with the S protein in vitro, the induced Th1 or Th2 cytokine levels were not significantly different upon re-stimulation with wild-type S or variant S, suggesting that the T-cell responses against the variants were the same as those against the wild-type virus. Upon Omicron XBB-challenge in hamsters, wild-type S-vaccination with Alhydrogel or AS03 reduced lung virus titers on Day 3, and the Quil-A adjuvanted group showed less body weight loss, although serum neutralizing antibody titers against XBB were barely detected in vitro. Collectively, recombinant vaccines coupled with different adjuvants may be promising modalities to combat new variant viruses by inducing various arms of the immune response.

Genomic analysis of SARS-CoV-2 in forensic autopsy cases of COVID-19.

Numerous genomic analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been conducted, highlighting its variations and lineage transitions. Despite the importance of forensic autopsy in investigating deaths due to coronavirus disease 2019 (COVID-19), including out-of-hospital deaths, viral genomic analysis has rarely been reported due in part to postmortem changes. In this study, various specimens were collected from 18 forensic autopsy cases with SARS-CoV-2 infection. Reverse-transcription quantitative polymerase chain reaction revealed the distribution of the virus in the body, primarily in the respiratory organs. Next-generation sequencing determined the complete genome sequences in 15 of the 18 cases, although some cases showed severe postmortem changes or degradation of tissue RNA. Intrahost genomic diversity of the virus was identified in one case of death due to COVID-19. The accumulation of single-nucleotide variations in the lung of the case suggested the intrahost evolution of SARS-CoV-2. Lung of the case showed diffuse alveolar damage histologically and positivity for SARS-CoV-2 by immunohistochemical analysis and in situ hybridization, indicating virus-associated pneumonia. This study provides insights into the feasibility of genomic analysis of SARS-CoV-2 in forensic autopsy cases and the potential for uncovering important information in COVID-19 deaths, including out-of-hospital deaths.

Organization of transcription and 3D genome as revealed by live-cell imaging.

Higher-order genomic structures play a critical role in regulating gene expression by influencing the spatial proximity of promoters and enhancers. Live-cell imaging studies have demonstrated that three-dimensional genome structures undergo dynamic changes over time. Transcription is also dynamic, with genes frequently switching between active and inactive states. Recent observations suggest that the formation of condensates, composed of transcription-related factors, RNA, and RNA-binding proteins, around genes can regulate transcription. Advancements in technology have facilitated the visualization of the intricate spatiotemporal relationship between higher-order genomic structures, condensate formation, and transcriptional activity in living cells.

Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryo.

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.