mRNA-encoded Cas13 treatment of Influenza via site-specific degradation of genomic RNA.

The CRISPR-Cas13 system has been proposed as an alternative treatment of viral infections. However, for this approach to be adopted as an antiviral, it must be optimized until levels of efficacy rival or exceed the performance of conventional approaches. To take steps toward this goal, we evaluated the influenza viral RNA degradation patterns resulting from the binding and enzymatic activity of mRNA-encoded LbuCas13a and two crRNAs from a prior study, targeting PB2 genomic and messenger RNA. We found that the genome targeting guide has the potential for significantly higher potency than originally detected, because degradation of the genomic RNA is not uniform across the PB2 segment, but it is augmented in proximity to the Cas13 binding site. The PB2 genome targeting guide exhibited high levels (>1 log) of RNA degradation when delivered 24 hours post-infection in vitro and maintained that level of degradation over time, with increasing multiplicity of infection (MOI), and across modern influenza H1N1 and H3N2 strains. Chemical modifications to guides with potent LbuCas13a function, resulted in nebulizer delivered efficacy (>1-2 log reduction in viral titer) in a hamster model of influenza (Influenza A/H1N1/California/04/09) infection given prophylactically or as a treatment (post-infection). Maximum efficacy was achieved with two doses, when administered both pre- and post-infection. This work provides evidence that mRNA-encoded Cas13a can effectively mitigate Influenza A infections opening the door to the development of a programmable approach to treating multiple respiratory infections.

Transcriptome profiles of organ tissues from pigs experimentally infected with African swine fever virus in early phase of infection.

African swine fever, caused by African swine fever virus (ASFV), is a highly contagious and fatal disease that poses a significant threat to the global pig industry. The limited information on ASFV pathogenesis and ASFV-host interactions has recently prompted numerous transcriptomic studies. However, most of these studies have focused on elucidating the transcriptome profiles of ASFV-infected porcine alveolar macrophages in vitro. Here, we analyzed dynamic transcriptional patterns in vivo in nine organ tissues (spleen, submandibular lymph node, mesenteric lymph node, inguinal lymph node, tonsils, lungs, liver, kidneys, and heart) obtained from pigs in the early stages of ASFV infection (1 and 3 d after viremia). We observed rapid spread of ASFV to the spleen after viremia, followed by broad transmission to the liver and lungs and subsequently, the submandibular and inguinal lymph nodes. Profound variations in gene expression patterns were observed across all organs and at all time-points, providing an understanding of the distinct defence strategies employed by each organ against ASFV infection. All ASFV-infected organs exhibited a collaborative response, activating immune-associated genes such as S100A8, thereby triggering a pro-inflammatory cytokine storm and interferon activation. Functional analysis suggested that ASFV exploits the PI3K-Akt signalling pathway to evade the host immune system. Overall, our findings provide leads into the mechanisms underlying pathogenesis and host immune responses in different organs during the early stages of infection, which can guide further explorations, aid the development of efficacious antiviral strategies against ASFV, and identify valuable candidate gene targets for vaccine development.

Amyloid-ß neuropathology induces bone loss in male mice by suppressing bone formation and enhancing bone resorption.

Alzheimer's disease (AD) and osteoporosis often coexist in the elderly. Although observational studies suggest an association between these two diseases, the pathophysiologic link between AD and skeletal health has been poorly defined. We examined the skeletal phenotype of 5xFAD mice, an AD model with accelerated neuron-specific amyloid-ß accumulation causing full-blown AD phenotype by the age of 8 months. Micro-computed tomography indicated significantly lower trabecular and cortical bone parameters in 8-month-old male, but not female, 5xFAD mice than sex-matched wild-type littermates. Dynamic histomorphometry revealed reduced bone formation and increased bone resorption, and quantitative RT-PCR showed elevated skeletal RANKL gene expression in 5xFAD males. These mice also had diminished body fat percentage with unaltered lean mass, as determined by dual-energy X-ray absorptiometry (DXA), and elevated Ucp1 mRNA levels in brown adipose tissue, consistent with increased sympathetic tone, which may contribute to the osteopenia observed in 5xFAD males. Nevertheless, no significant changes could be detected between male 5xFAD and wild-type littermates regarding the serum and skeletal concentrations of norepinephrine. Thus, brain-specific amyloid-ß pathology is associated with osteopenia and appears to affect both bone formation and bone resorption. Our findings shed new light on the pathophysiologic link between Alzheimer's disease and osteoporosis.

Antifungal activity of aminopyrrolnitrin against Trichophyton verrucosum in a guinea pig model of dermatophytosis.

BACKGROUND: Dermatophytosis is a common and major public health concern worldwide. Despite the increasing availability of antifungal drugs, relapses and untreated cases of dermatophyte infections are reported. Therefore, novel antifungal agents are required. Aminopyrrolnitrin (APRN) shows promise for dermatophytosis treatment because of its antifungal activity. OBJECTIVES: This study aimed to assess the antifungal properties of APRN against Trichophyton verrucosum (T. verrucosum), in both laboratory settings and a guinea pig model. METHODS: The minimum inhibitory concentrations (MICs) of APRN and enilconazole against T. verrucosum were determined according to the CLSI M38 method. The skins of 16 male guinea pigs were infected with 1.0 × 108 conidia of T. verrucosum and the animals were grouped into sets of four: negative control group (NC) received normal saline; positive control group (PC) received 2 µg/mL of enilconazole; and APRN4 and APRN8 received 4 and 8 µg/mL of APRN, respectively. Clinical, mycological and histological efficacies were measured after 10 days. RESULTS: The MIC90 of APRN and enilconazole against T. verrucosum was 4 and 2 µg/mL, respectively. The clinical scores of PC, APRN4, and APRN8 were significantly lower than those of NC. Clinical and mycological efficacies were higher for APRN8, APRN4 and PC. No fungi were observed in the skin tissues of APRN4 and APRN8, while fungi were observed in 50% of the PC. CONCLUSION: APRN showed antifungal activity against T. verrucosum in vitro and in vivo and is a potential candidate for the treatment of dermatophytosis.

Impact of rumen cannulation surgery on rumen microbiota composition in Hanwoo steers.

Rumen cannulation is a surgical technique used to collect rumen contents from ruminants. However, rumen cannulation surgery may potentially impact the composition of the rumen microbiota. This study aimed to examine the longitudinal alterations in the rumen microbiota composition of Hanwoo steers after cannulation surgery. In this study, eight Hanwoo steers were used; four steers underwent rumen cannulation surgery (cannulation group), while the remaining four were left intact (control group). Rumen samples were collected from all eight steers using the stomach tubing method on the day before surgery (day 0) and on postoperative days 1, 4, 7, 10, 14, 17, 21, 24, and 28, resulting in 80 samples (10 timepoints × 8 animals). The microbiota of all 80 samples were analyzed using 16S rRNA gene amplicon sequencing with Quantitative Insights into Microbial Ecology version 2 (QIIME2). There were no significant differences (p > 0.05) in all major phyla and most major genera representing at least 0.5% of total sequences across all 80 samples between the control and cannulation groups on the preoperative and postoperative days. However, while the alpha diversity indices did not differ (p > 0.05) between the two groups on the preoperative day, they significantly differed (p < 0.05) between the two groups on the postoperative days. Further, the overall microbial distribution based on both unweighted and weighted principal coordinate analysis plots significantly differed (p < 0.05) between the two groups on both the preoperative and postoperative days. Orthogonal polynomial contrasts indicated that major genera and microbial diversity in the cannulation group decreased following surgery but returned to their initial states by postoperative day 28. In conclusion, this study demonstrates that rumen cannulation surgery affects some major taxa and microbial diversity, suggesting that the rumen cannulation method can alter the composition of rumen microbiota in Hanwoo steers.

Abscisic acid signaling through LANCL2 and PPARγ induces activation of p38MAPK resulting in dormancy of prostate cancer metastatic cells.

Prostate cancer (PCa) is one the most common malignancies in men. The high incidence of bone metastasis years after primary therapy suggests that disseminated tumor cells must become dormant, but maintain their ability to proliferate in the bone marrow. Abscisic acid (ABA) is a stress response molecule best known for its regulation of seed germination, stomal opening, root shoot growth and other stress responses in plants. ABA is also synthesized by mammalian cells and has been linked to human disease. The aim of the present study was to examine the role of ABA in regulating tumor dormancy via signaling through lanthionine synthetase C­like protein 2 (LANCL2) and peroxisome proliferator activated receptor γ (PPARγ) receptors. ABA signaling in human PCa cell lines was studied using targeted gene knockdown (KD), western blotting, quantitative PCR, cell proliferation, migration, invasion and soft agar assays, as well as co­culture assays with bone marrow stromal cells. The data demonstrated that ABA signaling increased the expression of p21, p27 and p16, while inhibiting viability, migration, invasion and colony size in a reversable manner without toxicity. ABA also induced p38MAPK activation and NR2F1 signaling. Targeted gene KD of LANCL2 and PPARγ abrogated the cellular responses to ABA. Taken together, these data demonstrate that ABA may induce dormancy in PCa cell lines through LANCL2 and PPARγ signaling, and suggest novel targets to manage metastatic PCa growth.

Conversion of Host Cell Receptor into Virus Destructor by Immunodisc to Neutralize Diverse SARS-CoV-2 Variants.

The decreasing efficacy of antiviral drugs due to viral mutations highlights the challenge of developing a single agent targeting multiple strains. Using host cell viral receptors as competitive inhibitors is promising, but their low potency and membrane-bound nature have limited this strategy. In this study, the authors show that angiotensin-converting enzyme 2 (ACE2) in a planar membrane patch can effectively neutralize all tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that emerged during the COVID-19 pandemic. The ACE2-incorporated membrane patch implemented using nanodiscs replicated the spike-mediated membrane fusion process outside the host cell, resulting in virus lysis, extracellular RNA release, and potent antiviral activity. While neutralizing antibodies became ineffective as the SARS-CoV-2 evolved to better penetrate host cells the ACE2-incorporated nanodiscs became more potent, highlighting the advantages of using receptor-incorporated nanodiscs for antiviral purposes. ACE2-incorporated immunodisc, an Fc fusion nanodisc developed in this study, completely protected humanized mice infected with SARS-CoV-2 after prolonged retention in the airways. This study demonstrates that the incorporation of viral receptors into immunodisc transforms the entry gate into a potent virucide for all current and future variants, a concept that can be extended to different viruses.

Gut microbiota alteration with growth performance, histopathological lesions, and immune responses in Salmonella Typhimurium-infected weaned piglets.

Salmonella Typhimurium can cause gastroenteritis in weaned piglets, which are particularly vulnerable to dietary changes and dysfunction of their immature organs. The colonization of S. Typhimurium could disrupt the gut microbiota and increase susceptibility to the bacterium. This study aimed to investigate the alterations of gut microbiota in S. Typhimurium-infected weaned piglets. Ten 49-day-old pigs were divided into two groups: S. Typhimurium-inoculated (ST, n = 6) and negative control (NC, n = 4) groups. The body weight and S. Typhimurium fecal shedding were monitored for 14 days after S. Typhimurium inoculation (dpi). The intestinal tissues were collected at 14 dpi; histopathological lesions and cytokine gene expression were evaluated. The gut microbiome composition and short-chain fatty acid concentrations were analyzed in fecal samples collected at 14 dpi. The average daily gain and gut microbiota alpha diversity in ST group tended to be lower than NC group at 14 dpi. Linear discriminant analysis effect size results showed a significant increase in the abundance of two genera and five species, while a significant decrease was observed in the five genera and nine species within the gut microbiota of ST group. Among the significantly less abundant bacteria in the ST group, Lachnospira eligens and Anaerobium acetethylicum produce acetate and butyrate, and may be considered as key S. Typhimurium infection-preventing bacteria. The overall results provide invaluable information about changes in the gut microbiota of S. Typhimurium-infected weaned piglets, which can be used to develop alternative measures to antibiotics and prevent ST bacterial infection.

Mycoplasma hyorhinis infection promotes TNF-α signaling and SMAC mimetic-mediated apoptosis in human prostate cancer.

Growing evidence suggests an association between Mycoplasma infections and the development and progression of prostate cancer (PCa). In this study, we report that chronic and persistent M. hyorhinis infection induced robust TNF-α secretion from PCa cells. TNF-α secreted from M. hyorhinis-infected PCa cells subsequently led to activation of the NF-κB pathway. Chronic M. hyorhinis infection induced gene expression of pro-inflammatory cytokines and chemokines in a NF-κB-dependent manner and promoted cell proliferation, migration, and invasion in PCa cells. The elimination of M. hyorhinis in PCa cells significantly blocked TNF-α secretion, gene expression of cytokines and chemokines, migration, and invasion in PCa cells, suggesting M. hyorhinis-induced TNF-α plays an important role to promote malignant transformation of PCa. Furthermore, second mitochondria-derived activator of caspases (SMAC) mimetics potentiated caspase activation and cell death in M. hyorhinis-infected PCa by antagonizing inhibitor of apoptosis proteins (IAPs) activity. Tissue microarray analysis indicated that TNF-α is co-expressed in M. hyorhinis-infected human patient tissues. Findings from this study advance our understanding of the mycoplasma-oncogenesis process and suggest the potential for new approaches for preventions, diagnosis, and therapeutic approaches against prostate cancers.

Pathobiological analysis of african swine fever virus contact-exposed pigs and estimation of the basic reproduction number of the virus in Vietnam.

BACKGROUND: African swine fever (ASF), caused by African swine fever virus (ASFV), is a fatal disease affecting wild and domestic pigs. Since China reported the first ASF outbreak in August 2018, ASFV has swept over the neighbouring Asian countries. However, studies involving experimental pig-to-pig ASFV transmission in Vietnam are lacking. The main objective of this experimental study was to demonstrate the pathobiological characteristics of ASFV contact-exposed pigs and estimate their basic reproduction number (R0) in Vietnam. Fifteen pigs were randomly divided into two groups: experimental (n = 10) and negative control (n = 5) groups. One pig in the experimental group was intramuscularly inoculated with ASFV strain from Vietnam in 2020 and housed with the uninoculated pigs during the study period (28 days). RESULTS: The inoculated pig died 6 days post-inoculation, and the final survival rate was 90.0%. We started observing viremia and excretion of ASFV 10 days post-exposure in contact-exposed pigs. Unlike the surviving and negative control pigs, all necropsied pigs showed severe congestive splenomegaly and moderate-to-severe haemorrhagic lesions in the lymph nodes. The surviving pig presented with mild haemorrhagic lesions in the spleen and kidneys. We used Susceptible-Infectious-Removed models for estimating R0. The R0 values for exponential growth (EG) and maximum likelihood (ML) were calculated to be 2.916 and 4.015, respectively. In addition, the transmission rates (ß) were estimated to be 0.729 (95% confidence interval [CI]: 0.379-1.765) for EG and 1.004 (95% CI: 0.283-2.450) for ML. CONCLUSIONS: This study revealed pathobiological and epidemiological information in about pig-to-pig ASFV transmission. Our findings suggested that culling infected herds within a brief period of time may mitigate the spread of ASF outbreaks.

Raw potato starch diet supplement in weaned pigs could reduce Salmonella Typhimurium infection by altering microbiome composition and improving immune status.

Backgorund: Salmonella enterica serovar Typhimurium (ST) is one of the causative agents of gastroenteritis in pigs. Pigs fed a diet supplemented with raw potato starch (RPS) have improved gut health by the alteration of the microbiota composition and production of short-chain fatty acids (SCFAs). This study aimed to evaluate the effects of RPS supplementation in reducing infection severity and fecal shedding in ST-infected pigs. Methods: The weaned experimental pigs were divided into two groups: CON (n = 6) fed a corn/soybean-based diet and TRT (n = 6) supplemented with 5% RPS. After 21 d, the pigs were inoculated with ST, and their body weight, clinical signs, and fecal shedding of ST were monitored for 14 d. At 14 d post-inoculation (dpi), the jejunum, cecum, ileum, and colon tissues were collected from euthanized pigs, and histopathological lesions and cytokine gene expression were compared. Additionally, blood samples at 2 dpi were analyzed for gene ontology enrichment. Moreover, the gutmicrobiome was analyzed using 16S rRNA metagenomic sequencing, and the SCFA concentration was measured using gas chromatography. Results: The average daily weight gain was significantly higher in TRT than in CON during the ST infection period; however, histopathological lesion scores were significantly lower in TRT than in CON. The relative abundance of nine genera of butyrate- and acetate-producing bacteria significantly increased in TRT compared with that of only two acetate-producing bacteria in CON. Among the genes involved in the immune response, IL-18 expression level was significantly lower in the jejunum and colon in TRT than in CON. Furthermore, Reg3γ expression was significantly different in the cecum and colon of both groups. Conclusion: The diet supplemented with RPS in weaned pigs could result in predominance of butyrate- and acetate-producing bacteria, reducing the severity of ST infection by improving the immune status.

Multi-organ metastases of primary chondrosarcoma of the mammary gland of a spayed Toy Poodle.

We report the clinical and histopathological findings associated with multi-organ metastases of primary mammary chondrosarcoma in a 12-year-old spayed female Toy Poodle. At post-mortem examination, multifocal, sharply demarcated, grey-white to bright brown, round nodules of variable size were randomly distributed in the lungs, myocardium, liver, pancreas, spleen, intestinal tract and kidneys. Histologically, immature cartilage structures and primitive mesenchymal cells were seen in these organs. Neoplastic cells located in the cartilaginous basophilic extracellular matrix had cytoplasmic vacuolation and round vesicular nuclei and were stained with Safranin O and Alcian blue. To the best of our knowledge, this is the first report of a multi-organ metastatic chondrosarcoma that originated in the mammary gland of a dog.

Fingolimod mitigates memory loss in a mouse model of Gulf War Illness amid decreasing the activation of microglia, protein kinase R, and NFκB.

Gulf War Illness (GWI) is an unrelenting multi-symptom illness with chronic central nervous system and peripheral pathology affecting veterans from the 1991 Gulf War and for which effective treatment is lacking. An increasing number of studies indicate that persistent neuroinflammation is likely the underlying cause of cognitive and mood dysfunction that affects veterans with GWI. We have previously reported that fingolimod, a drug approved for the treatment of relapsing-remitting multiple sclerosis, decreases neuroinflammation and improves cognition in a mouse model of Alzheimer's disease. In this study, we investigated the effect of fingolimod treatment on cognition and neuroinflammation in a mouse model of GWI. We exposed C57BL/6 J male mice to GWI-related chemicals pyridostigmine bromide, DEET, and permethrin, and to mild restraint stress for 28 days (GWI mice). Control mice were exposed to the chemicals' vehicle only. Starting 3 months post-exposure, half of the GWI mice and control mice were orally treated with fingolimod (1 mg/kg/day) for 1 month, and the other half were left untreated. Decreased memory on the Morris water maze test was detected in GWI mice compared to control mice and was reversed by fingolimod treatment. Immunohistochemical analysis of brain sections with antibodies to Iba1 and GFAP revealed that GWI mice had increased microglia activation in the hippocampal dentate gyrus, but no difference in reactive astrocytes was detected. The increased activation of microglia in GWI mice was decreased to the level in control mice by treatment with fingolimod. No effect of fingolimod treatment on gliosis in control mice was detected. To explore the signaling pathways by which decreased memory and increased neuroinflammation in GWI may be protected by fingolimod, we investigated the involvement of the inflammatory signaling pathways of protein kinase R (PKR) in the cerebral cortex of these mice. We found increased phosphorylation of PKR in the brain of GWI mice compared to controls, as well as increased phosphorylation of its most recognized downstream effectors: the α subunit of eukaryotic initiation factor 2 (eIF2α), IκB kinase (IKK), and the p65 subunit of nuclear factor-κB (NFκB-p65). Furthermore, we found that the increased phosphorylation level of these three proteins were suppressed in GWI mice treated with fingolimod. These results suggest that activation of PKR and NFκB signaling may be important for the regulation of cognition and neuroinflammation in the GWI condition and that fingolimod, a drug already approved for human use, may be a potential candidate for the treatment of GWI.

Synthesis and physicochemical characterization of acyl myricetins as potential anti-neuroexocytotic agents.

Acyl myricetins (monopropionyl-, dipropionyl-, and monooctanoyl-myricetin, termed as MP1, MP2, and MO1, respectively) were synthesized through enzymatic or non-enzymatic esterification reaction of myricetin aglycone. Structure study indicated the hydroxyl group at C4' in B-ring was highly susceptible to acylation. Over its parental myricetin, acylated compounds showed enhanced lipophilicity (from 7.4- to 26.3-fold) and oxidative stability (from 1.9- to 3.1-fold) on the basis of logP and decay rate, respectively. MO1, presenting the physicochemical superiority compared to the others, provided lowest EC50 value of 2.51 µM on inhibition of neutrotransmitter release and CC50 value of 59.0 µM, leading to widest therapeutic window. All myricetin esters did not show any irritation toxicity when assessed with a chicken embryo assay. This study describes information on acylation of myricetin that has not yet been explored, and suggests that MO1 has membrane fusion-arresting and anti-neuroexocytotic potential for industrial application due to its enhanced biological properties.

HER2 as a potential therapeutic target on quiescent prostate cancer cells.

Quiescent prostate cancer (PCa) cells are common in tumors but are often resistant to chemotherapy. Quiescent PCa cells are also enriched for a stem-like tumor initiating population, and can lead to recurrence after dormancy. Unfortunately, quiescent PCa cells are difficult to identify and / or target with treatment in part because the relevant markers are intracellular and regulated by protein stability. We addressed this problem by utilizing PCa cells expressing fluorescent markers for CDKN1B (p27) and CDT1, which can separate viable PCa cells into G0, G1, or combined S/G2/M populations. We used FACS to collect G1 and G0 PC3 PCa cells, isolated membrane proteins, and analyzed protein abundance in G0 vs G1 cells by gas chromatography mass spectrometry. Enrichment analysis identified nucleocytoplasmic transport as the most significantly different pathway. To identify cell surface proteins potentially identifying quiescent PCa cells for future patient samples or for antibody based therapeutic research, we focused on differentially abundant plasma membrane proteins, and identified ERBB2 (HER2) as a cell surface protein enriched on G0 PCa cells. High HER2 on the cell membrane is associated with quiescence in PCa cells and likely induced by the bone microenvironment. Using a drug conjugated anti-HER2 antibody (trastuzumab emtansine) in a mouse PCa xenograft model delayed metastatic tumor growth, suggesting approaches that target HER2-high cells may be beneficial in treating PCa. We propose that HER2 is deserving of further study in PCa as a target on quiescent cells to prevent recurrence, decrease chemotherapy resistance, or eradicate minimal residual disease.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean Red Ginseng.

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

Dysregulation of sphingosine-1-phosphate (S1P) and S1P receptor 1 signaling in the 5xFAD mouse model of Alzheimer's disease.

Sphingolipid-1-phosphate (S1P) signaling through the activation S1P receptors (S1PRs) plays critical roles in cellular events in the brain. Aberrant S1P metabolism has been identified in the brains of Alzheimer's disease (AD) patients. Our recent studies have shown that treatment with fingolimod, an analog of sphingosine, provides neuroprotective effects in five familiar Alzheimer disease (5xFAD) transgenic mice, resulting in the reduction of amyloid-ß (Aß) neurotoxicity, inhibition of activation of microglia and astrocytes, increased hippocampal neurogenesis, and improved learning and memory. However, the pathways by which dysfunctional S1P and S1PR signaling may associate with the development of AD-like pathology remain unknown. In this study, we investigated the alteration of signaling of S1P/S1P receptor 1 (S1PR1), the most abundant S1PR subtype in the brain, in the cortex of 5xFAD transgenic mice at 3, 8, and 14 months of age. Compared to non-transgenic wildtype (WT) littermates, we found significant decreased levels of sphingosine kinases (SphKs), increased S1P lyase (S1PL), and increased S1PR1 in 8- and 14-month-old, but not in 3-month-old 5xFAD mice. Furthermore, we detected increased activation of the S1PR1 downstream pathway of Akt/mTor/Tau signaling in aging 5xFAD mice. Treatment with fingolimod from 1 to 8 months of age reversed the levels of SphKs, S1PL, and furthermore, those of S1PR1 and its downstream pathway of Akt/mTor/Tau signaling. Together the data reveal that dysregulation of S1P and S1PR signaling may associate with the development of AD-like pathology through Akt/mTor/Tau signaling.

Johne's disease in two Korean black goats (Capra hircus coreanae) with clinical and subclinical infection: A case report.

Johne's disease (JD) is a chronic enteric infection in ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). JD infection is more difficult to diagnose in goats than cattle because MAP can insidiously affect small ruminants. Few reports have used pathological and molecular diagnosis for cases in Korean black goats. Here, we present our results from two MAP-infected goats. Case 1 was categorized as clinically significant (stage IV), with severe clinical signs and a high antibody titer (S/P ratio, 158.9%). Case 2 was in the subclinical stage (stage II); however, the goat suddenly died without any clinical signs (S/P ratio, 70.9%). DNA from the organ tissues and feces from Case 1 showed a strong positive PCR result for MAP, whereas Case 2 only exhibited a very weak reaction in the fecal sample. Moreover, fecal DNA from both cases was genotyped as C-type MAP using the PCR-REA method. Gastrointestinal organ tissues (jejunum, ileum, colon, and mesenteric lymph nodes) from Case 1 showed moderate-to-severe lesions, and acid-fast bacilli were observed. In contrast, Case 2 showed intact-to-mild pathological lesions, and acid-fast bacilli were detected in the colon, mesenteric lymph nodes, and liver. In addition, Case 2 was co-infected with Corynebacterium pseudotuberculosis, which caused caseous lymphadenitis. This case study provides valuable information regarding the pathological and molecular characteristics of JD-infected Korean black goats. The results highlighted the differences in pathological lesions between clinically and subclinically infected goats, which help veterinarians to develop better strategies to control MAP in goat farms.

Nebulized mRNA-Encoded Antibodies Protect Hamsters from SARS-CoV-2 Infection.

Despite the success of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines, there remains a clear need for new classes of preventatives for respiratory viral infections due to vaccine hesitancy, lack of sterilizing immunity, and for at-risk patient populations, including the immunocompromised. While many neutralizing antibodies have been identified, and several approved, to treat COVID-19, systemic delivery, large doses, and high costs have the potential to limit their widespread use, especially in low- and middle-income countries. To use these antibodies more efficiently, an inhalable formulation is developed that allows for the expression of mRNA-encoded, membrane-anchored neutralizing antibodies in the lung to mitigate SARS-CoV-2 infections. First, the ability of mRNA-encoded, membrane-anchored, anti-SARS-CoV-2 antibodies to prevent infections in vitro is demonstrated. Next, it is demonstrated that nebulizer-based delivery of these mRNA-expressed neutralizing antibodies potently abrogates disease in the hamster model. Overall, these results support the use of nebulizer-based mRNA expression of neutralizing antibodies as a new paradigm for mitigating respiratory virus infections.