Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.

The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.

Proteomic analysis reveals that aging rabbit vocal folds are more vulnerable to changes caused by systemic dehydration.

BACKGROUND: Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol. RESULTS: Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of - 5.4% and - 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration. CONCLUSIONS: Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

Quantification of murine myocardial infarct size using 2-D and 4-D high-frequency ultrasound.

Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via two-dimensional (2-D) echocardiographic akinetic length and four-dimensional (4-D) echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2-D and 4-D echocardiography. Infarct size established via histology was compared with ultrasound-based metrics via linear regression analysis. Two-dimensional echocardiographic akinetic length (r = 0.76, P = 0.03), 4-D echocardiographic infarct volume (r = 0.85, P = 0.008), and surface area (r = 0.90, P = 0.002) correlate well with histology. Although both 2-D and 4-D echocardiography were reliable measurement techniques to assess infarct, 4-D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4-D data also provides additional infarct sizing techniques, which correlate with histology (surface strain: r = 0.94, P < 0.001, transmural thickness: r = 0.76, P = 0.001). Two-dimensional echocardiographic akinetic length, 4-D echocardiography ultrasound, and strain provide effective in vivo methods for measuring fibrotic scarring after MI.NEW & NOTEWORTHY Our study supports that both 2-D and 4-D echocardiographic analysis techniques are reliable in quantifying infarct size though 4-D ultrasound provides a more holistic image of LV function and structure, especially after myocardial infarction. Furthermore, 4-D strain analysis correctly identifies infarct size and regional LV dysfunction after MI. Therefore, these techniques can improve functional insight into the impact of pharmacological interventions on the pathophysiology of cardiac disease.

Canary Seed (Phalaris canariensis L.) Peptides Prevent Obesity and Glucose Intolerance in Mice Fed a Western Diet.

Previous research showed that canary seed (Phalaris canariensis L.) peptides (CSP) possess robust in vitro antiobesity properties via inhibition of pancreatic lipase (PL). Nevertheless, no studies have yet explored their antiobesity properties in vivo. Consequently, we investigated the effects of CSP in C57BL/6J mice under a Western diet (WD). Mice were assigned into groups and fed a normal diet (ND) or a WD accompanied by an oral dose of CSP (250 or 500 mg/kg/day), orlistat (40 mg/kg/day), or distilled water. The results showed that consuming CSP can provide metabolic benefits, including preventing weight gain by up to 20%, increasing glucose tolerance, and reducing insulin, leptin, and LDL/VLDL levels in plasma. Conversely, total ghrelin was unaffected by CSP-500, but decreased by CSP-250, and amplified by orlistat. Surprisingly, CSP-250 was more effective in preventing weight gain and promoting satiety than CSP-500. Parallel to this, protein absorption in CSP-500 was decreased, supported by a rise in fecal crude protein (+3.5%). Similarly, fecal fat was increased by orlistat (38%) and was unaffected by CSP-250 (3.0%) and CSP (3.0%), comparatively to WD (2.5%). Despite this, both CSP treatments were equally effective in decreasing hepatic steatosis and avoiding hyperlipidemia. Furthermore, the enzymatic analysis showed that CSP-PL complexes dissociated faster (15 min) than orlistat-PL complexes (41 min). Lastly, CSP did not affect expression of hepatic lipid oxidation genes ACO and PPAR-α, but reduced the expression of the hydrolase gene LPL, and lipogenesis related genes FAS and ACC. Taken together, these results suggest that CSP antiobesity mechanism relies on lipid metabolism retardation to increase fat transit time and subsequently suppress hunger.

Listeria adhesion protein-expressing bioengineered probiotics prevent fetoplacental transmission of Listeria monocytogenes in a pregnant Guinea pig model.

Pregnancy is a high-risk factor for foodborne pathogen Listeria monocytogenes (Lm), which causes abortion, premature birth, or stillbirth. The primary route of Lm transmission is oral hence intestinal epithelial barrier crossing is a prerequisite for systemic spread. Intestinal barrier crossing, in part, is attributed to the interaction of Listeria adhesion protein (LAP) with its cognate receptor, Hsp60. In a recent study, we showed that oral-dosing of bioengineered Lactobacillus caseiprobiotic (BLP) expressing the LAP protected nonpregnant mice from lethal infection; however, its ability to prevent listeriosis during pregnancy is not known. Therefore, we investigated whether BLP could prevent fetoplacental transmission of Lm in a pregnant guinea pig model. After 14 consecutive days on probiotic (~109 CFU/ml in drinking water), pregnant guinea pigs (gestational days 24-28) were orally challenged with Lm (9 × 108-2.5 × 109 CFU/animal) and were euthanized 72 h post-infection. Maternal mesenteric lymph node (MLN), liver, spleen, lungs, blood, and placenta, and fetal liver were analyzed for the presence/absence of Lm. All tissues/organs from Lm-challenged naïve dams and fetuses were Lm positive. Similar tissue distribution was also seen in guinea pigs that received wild-type Lactobacillus casei (LbcWT). Remarkably, Lm was absent in the maternal blood, kidney, lungs, and placenta, and fetal liver from the BLP-fed group even though the Lm was present in the maternal liver, spleen, and MLN. BLP feeding also suppressed Lm-induced inflammatory response in mothers. These data highlight the potential for the prevention of fetoplacental transmission of Lm by LAP-expressing BLP during pregnancy.

RNA sequencing identifies transcriptional changes in the rabbit larynx in response to low humidity challenge.

BACKGROUND: Voice disorders are a worldwide problem impacting human health, particularly for occupational voice users. Avoidance of surface dehydration is commonly prescribed as a protective factor against the development of dysphonia. The available literature inconclusively supports this practice and a biological mechanism for how surface dehydration of the laryngeal tissue affects voice has not been described. In this study, we used an in vivo male New Zealand white rabbit model to elucidate biological changes based on gene expression within the vocal folds from surface dehydration. Surface dehydration was induced by exposure to low humidity air (18.6% + 4.3%) for 8 h. Exposure to moderate humidity (43.0% + 4.3%) served as the control condition. Ilumina-based RNA sequencing was performed and used for transcriptome analysis with validation by RT-qPCR. RESULTS: There were 103 statistically significant differentially expressed genes identified through Cuffdiff with 61 genes meeting significance by both false discovery rate and fold change. Functional annotation enrichment and predicted protein interaction mapping showed enrichment of various loci, including cellular stress and inflammatory response, ciliary function, and keratinocyte development. Eight genes were selected for RT-qPCR validation. Matrix metalloproteinase 12 (MMP12) and macrophage cationic peptide 1 (MCP1) were significantly upregulated and an epithelial chloride channel protein (ECCP) was significantly downregulated after surface dehydration by RNA-Seq and RT-qPCR. Suprabasin (SPBN) and zinc activated cationic channel (ZACN) were marginally, but non-significantly down- and upregulated as evidenced by RT-qPCR, respectively. CONCLUSIONS: The data together support the notion that surface dehydration induces physiological changes in the vocal folds and justifies targeted analysis to further explore the underlying biology of compensatory fluid/ion flux and inflammatory mediators in response to airway surface dehydration.

Ductular and proliferative response of esophageal submucosal glands in a porcine model of esophageal injury and repair.

Esophageal injury is a risk factor for diseases such as Barrett's esophagus (BE) and esophageal adenocarcinoma. To improve understanding of signaling pathways associated with both normal and abnormal repair, animal models are needed. Traditional rodent models of esophageal repair are limited by the absence of esophageal submucosal glands (ESMGs), which are present in the human esophagus. Previously, we identified acinar ductal metaplasia in human ESMGs in association with both esophageal injury and cancer. In addition, the SOX9 transcription factor has been associated with generation of columnar epithelium and the pathogenesis of BE and is present in ESMGs. To test our hypothesis that ESMGs activate after esophageal injury with an increase in proliferation, generation of a ductal phenotype, and expression of SOX9, we developed a porcine model of esophageal injury and repair using radiofrequency ablation (RFA). The porcine esophagus contains ESMGs, and RFA produces a consistent and reproducible mucosal injury in the esophagus. Here we present a temporal assessment of this model of esophageal repair. Porcine esophagus was evaluated at 0, 6, 18, 24, 48, and 72 h and 5 and 7 days following RFA and compared with control uninjured esophagus. Following RFA, ESMGs demonstrated an increase in ductal phenotype, echoing our prior studies in humans. Proliferation increased in both squamous epithelium and ESMGs postinjury with a prominent population of SOX9-positive cells in ESMGs postinjury. This model promises to be useful in future experiments evaluating mechanisms of esophageal repair.NEW & NOTEWORTHY A novel porcine model of injury and repair using radiofrequency ablation has been developed, allowing for reproducible injury to the esophagus to study repair in an animal model with esophageal submucosal glands, a key anatomical feature and missing in rodent models but possibly harboring progenitor cells. There is a strong translational component to this porcine model given the anatomical and physiological similarities between pigs and humans.

Cell aggregation mediated by ACE2 deletion in Candida auris modulates fungal colonization and host immune responses in the skin.

Candida auris is an emerging multi-drug-resistant fungal pathogen that colonizes the skin and causes invasive infections in hospitalized patients. Multi-cellular aggregative phenotype is widely reported in the C. auris isolates, but its role in skin colonization and host immune response is not yet known. In this study, we generated aggregative phenotype by deleting the ACE2 gene in C. auris and determined the fungal colonization and host immune response using an intradermal mouse model of C. auris skin infection. Our results indicate that mice infected with ace2Δ strain had significantly lower fungal load after 3 and 14 days post-infections compared to the non-aggregative wild-type and the ACE2 reintegrated strain. The colonization of ace2Δ is associated with increased recruitment of CD11b+ Ly6G+ neutrophils and decreased accumulation of CD11b+ Ly6 Chi inflammatory monocytes and CD11b+ MHCII+ CD64+ macrophages. Furthermore, Th17 cells and type 3 innate lymphoid cells (ILCs) were significantly increased in the skin tissue of ace2Δ infected mice. Our findings suggest that aggregative phenotype mediated by ACE2 deletion in C. auris induces potent neutrophil and IL-17-mediated immune response and reduces fungal colonization in the skin.IMPORTANCEC. auris is a rapidly emerging fungal pathogen that can colonize hospitalized patients, especially in skin tissue, and cause invasive infections. C. auris isolates exhibit morphological heterogeneity, and the multicellular aggregative phenotype of C. auris is reported frequently in clinical settings. Understanding the role of fungal morphotypes in colonization, persistence, and immune response in the skin microenvironment will have potential applications in clinical diagnosis and novel preventive and therapeutic measures. Here, we utilized the murine model of intradermal infection and determined that the aggregative phenotype of C. auris as the result of ACE2 gene deletion elicits potential innate and adaptive immune responses in mice. These observations will help explain the differences in the skin colonization and immune responses of the aggregative morphotype of C. auris and open the door to developing novel antifungal therapeutics.

Yeast and filamentous Candida auris stimulate distinct immune responses in the skin.

Candida auris, an emerging multidrug-resistant fungal pathogen, predominately colonizes the human skin long term leading to subsequent life-threatening invasive infections. Fungal morphology is believed to play a critical role in modulating mucocutaneous antifungal immunity. In this study, we used an intradermal mouse model of C. auris infection to examine fungal colonization and the associated innate and adaptive immune response to yeast and filamentous C. auris strains. Our results indicate that mice infected with a filamentous C. auris had significantly decreased fungal load compared to mice infected with the yeast form. Mice infected with yeast and filamentous forms of C. auris stimulated distinct innate immune responses. Phagocytic cells (CD11b+Ly6G+ neutrophils, CD11b+Ly6Chi inflammatory monocytes, and CD11b+MHCII+CD64+ macrophages) were differentially recruited to mouse skin tissue infected with yeast and filamentous C. auris. The percentage and absolute number of interleukin 17 (IL-17) producing innate lymphoid cells, TCRγδ+, and CD4+ T cells in the skin tissue of mice infected with filamentous C. auris were significantly increased compared to the wild-type of yeast strain. Furthermore, complementation of filamentous mutant strain of C. auris (Δelm1 + ELM1) strain exhibited wild-type yeast morphology in vivo and induced comparable level of skin immune responses similar to mice infected with yeast strain. Collectively, our findings indicate that yeast and filamentous C. auris induce distinct local immune responses in the skin. The decreased fungal load observed in mouse skin infected with filamentous C. auris is associated with a potent IL-17 immune response induced by this morphotype.IMPORTANCECandida auris is a globally emerging fungal pathogen that transmits among individuals in hospitals and nursing home residents. Unlike other Candida species, C. auris predominantly colonizes and persists in skin tissue resulting in outbreaks of systemic infections. Understanding the factors that regulate C. auris skin colonization and host immune response is critical to develop novel preventive and therapeutic approaches against this emerging pathogen. We identified that yeast and filamentous forms of C. auris induce distinct skin immune responses in the skin. These findings may help explain the differential colonization and persistence of C. auris morphotypes in skin tissue. Understanding the skin immune responses induced by yeast and filamentous C. auris is important to develop novel vaccine strategies to combat this emerging fungal pathogen.

Generating prophylactic immunity against arboviruses in vertebrates and invertebrates.

The World Health Organization recently declared a global initiative to control arboviral diseases. These are mainly caused by pathogenic flaviviruses (such as dengue, yellow fever and Zika viruses) and alphaviruses (such as chikungunya and Venezuelan equine encephalitis viruses). Vaccines represent key interventions for these viruses, with licensed human and/or veterinary vaccines being available for several members of both genera. However, a hurdle for the licensing of new vaccines is the epidemic nature of many arboviruses, which presents logistical challenges for phase III efficacy trials. Furthermore, our ability to predict or measure the post-vaccination immune responses that are sufficient for subclinical outcomes post-infection is limited. Given that arboviruses are also subject to control by the immune system of their insect vectors, several approaches are now emerging that aim to augment antiviral immunity in mosquitoes, including Wolbachia infection, transgenic mosquitoes, insect-specific viruses and paratransgenesis. In this Review, we discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases.

Water Versus Electrolyte Rehydration on Vocal Fold Osmotic and Oxidative Stress Gene Expression.

OBJECTIVES: Systemic dehydration may induce osmotic and oxidative stress in the vocal folds, but our knowledge of the biology and mitigation with rehydration is limited. The purpose of this experiment was to evaluate whether systemic dehydration induces vocal fold oxidative and osmotic stress and to compare the impact of rehydration by water intake versus electrolyte intake on osmotic and oxidative stress-related gene expression. METHODS: Four-month-old male Sprague-Dawley rats (N = 32) underwent water restriction. Rehydration was achieved with ad libitum access to water or electrolytes for 24 hours. Rats were divided into four groups: euhydration control, dehydration-only, dehydration followed by either water or electrolyte rehydration (n = 8/group). Gene expression was assessed via RT2 Gene Expression Profiler arrays. RESULTS: With respect to oxidative stress, 10 genes were upregulated and 2 were downregulated after vocal fold dehydration compared with the euhydrated control. Concerning osmotic stress, six genes were upregulated with dehydration only, six genes were upregulated following rehydration with water, whereas a single gene was upregulated with electrolyte rehydration. All genes with significantly different expression between the rehydration groups showed lower expression with electrolytes compared with water. CONCLUSIONS: The results support a potential role of oxidative and osmotic stresses in vocal folds related to systemic dehydration. The differences in stress-related gene expression in vocal fold tissue between rehydration with electrolytes or water, albeit modest, suggest that both rehydration options offer clinical utility to subjects experiencing vocal fold dehydration with preliminary evidence that electrolytes may be more effective than water in resolving osmotic stress. LEVEL OF EVIDENCE: NA (prospective animal study) Laryngoscope, 134:4636-4641, 2024.

In Vivo Visualization and Quantification of Rat Laryngeal Blood Supply After Hydration Challenge.

OBJECTIVES: Systemic dehydration decreases total body blood volume; however, hemodynamic alterations at the level of local organs, such as the larynx, remain unclear. Here we sought to quantify superior thyroid artery (STA) blood flow after dehydration and rehydration using in vivo magnetic resonance angiography (MRA) and ultrasound imaging in a rat model. METHODS: Male Sprague-Dawley rats (N = 17) were included in this prospective, repeated measures design. Rats first underwent MRA to determine baseline STA cross-sectional area, followed by high-frequency in vivo ultrasound imaging to measure STA blood velocity at baseline. Next, rats were systemically dehydrated (water withholding), followed by rehydration (water ad-lib). Ultrasound imaging was repeated immediately after dehydration and following rehydration. The STA blood velocity and STA cross-sectional area were used to compute STA blood flow. Three rats served as temporal controls for ultrasound imaging. To determine if the challenges to hydration status affected the STA cross-sectional area, four rats underwent only MRA at baseline, dehydration, and rehydration. RESULTS: Systemic dehydration resulted in 10.5% average body weight loss. Rehydration resulted in average body weight gain of 10.9%. Statistically significant reductions were observed in STA mean blood flow rate after dehydration. Rehydration reversed these changes to pre-dehydration levels. No significant differences were observed in STA cross-sectional area with dehydration or rehydration. CONCLUSION: Systemic dehydration decreased blood flow in the superior thyroid artery. Rehydration restored blood flow in the STA. Change in hydration status did not alter the STA cross-sectional area. These preliminary findings demonstrate the feasibility of using ultrasound and MRA to quantify hemodynamic changes and visualize laryngeal blood vessels. LEVEL OF EVIDENCE: NA Laryngoscope, 134:779-785, 2024.

Scaffold-Forming Collagen and Motor-Endplate Expressing Muscle Cells for Porcine Laryngoplasty.

OBJECTIVE: Vocal fold paralysis impairs quality of life, and no curative injectable therapy exists. We evaluated injection of a novel in situ polymerizing (scaffold-forming) collagen in the presence and absence of muscle-derived motor-endplate expressing cells (MEEs) to promote medialization and recurrent laryngeal nerve (RLN) regeneration in a porcine model of unilateral vocal fold paralysis. METHODS: Twelve Yucatan minipigs underwent right RLN transection. Autologous muscle progenitor cells were isolated from muscle biopsies, differentiated, and induced to MEEs. Three weeks after RLN injury, animals received injections of collagen, collagen containing MEEs, or saline into the paralyzed right vocal fold. Stimulated laryngeal electromyography and acoustic vocalization were used for function assessments. Larynges were harvested and underwent histologic, gene expression, and further quantitative analyses. RESULTS: Injections were well-tolerated, with the collagen scaffold showing immunotolerance and collagen-encapsulated MEEs remaining viable. Collagen-treated paralyzed vocal folds showed increased laryngeal adductor muscle volumes relative to that of the uninjured side, with those receiving MEEs and collagen showing the highest volumes. Muscles injected with MEEs and collagen demonstrated increased expression of select neurotrophic (BDNF and NTN1), motor-endplate (DOK7, CHRNA1, and MUSK), and myogenic (MYOG and MYOD) related genes relative to saline controls. CONCLUSION: In a porcine model of unilateral vocal fold paralysis, injection of in situ polymerizing collagen in the absence and presence of MEEs enhanced laryngeal adductor muscle volume, modulated expression of neurotrophic and myogenic factors, and avoided adverse material-mediated immune responses. Further study is needed to determine long-term functional outcomes with this novel therapeutic approach. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

α-Gal Nanoparticles in CNS Trauma: II. Immunomodulation Following Spinal Cord Injury (SCI) Improves Functional Outcomes.

BACKGROUND: Previous investigations have shown that local application of nanoparticles presenting the carbohydrate moiety galactose-α-1,3-galactose (α-gal epitopes) enhance wound healing by activating the complement system and recruiting pro-healing macrophages to the injury site. Our companion in vitro paper suggest α-gal epitopes can similarly recruit and polarize human microglia toward a pro-healing phenotype. In this continuation study, we investigate the in vivo implications of α-gal nanoparticle administration directly to the injured spinal cord. METHODS: α-Gal knock-out (KO) mice subjected to spinal cord crush were injected either with saline (control) or with α-gal nanoparticles immediately following injury. Animals were assessed longitudinally with neurobehavioral and histological endpoints. RESULTS: Mice injected with α-gal nanoparticles showed increased recruitment of anti-inflammatory macrophages to the injection site in conjunction with increased production of anti-inflammatory markers and a reduction in apoptosis. Further, the treated group showed increased axonal infiltration into the lesion, a reduction in reactive astrocyte populations and increased angiogenesis. These results translated into improved sensorimotor metrics versus the control group. CONCLUSIONS: Application of α-gal nanoparticles after spinal cord injury (SCI) induces a pro-healing inflammatory response resulting in neuroprotection, improved axonal ingrowth into the lesion and enhanced sensorimotor recovery. The data shows α-gal nanoparticles may be a promising avenue for further study in CNS trauma.

Design of a Fibroblast Activation Protein-Targeted Radiopharmaceutical Therapy with High Tumor-to-Healthy-Tissue Ratios.

Because of upregulated expression on cancer-associated fibroblasts, fibroblast activation protein (FAP) has emerged as an attractive biomarker for the imaging and therapy of solid tumors. Although many FAP ligands have already been developed for radiopharmaceutical therapies (RPTs), most suffer from inadequate tumor uptake, insufficient tumor residence times, or off-target accumulation in healthy tissues, suggesting a need for further improvements. Methods: A new FAP-targeted RPT with a novel ligand (FAP8-PEG3-IP-DOTA) was designed by combining the desirable features of several previous ligand-targeted RPTs. Uptake and retention of [111In]In or [177Lu]Lu-FAP8-PEG3-IP-DOTA were assessed in KB, HT29, MDA-MB-231, and 4T1 murine tumor models by radioimaging or ex vivo biodistribution analyses. Radiotherapeutic potencies and gross toxicities were also investigated by monitoring tumor growth, body weight, and tissue damage in tumor-bearing mice. Results: FAP8-PEG3-IP-DOTA exhibited high affinity (half-maximal inhibitory concentration, 1.6 nM) and good selectivity for FAP relative to its closest homologs, prolyl oligopeptidase (half-maximal inhibitory concentration, ∼14.0 nM) and dipeptidyl peptidase-IV (half-maximal inhibitory concentration, ∼860 nM). SPECT/CT scans exhibited high retention in 2 different solid tumor models and minimal uptake in healthy tissues. Quantitative biodistribution analyses revealed tumor-to-healthy-tissue ratios of more than 5 times for all major organs, and live animal studies demonstrated 65%-93% suppression of tumor growth in all 4 models tested, with minimal or no evidence of systemic toxicity. Conclusion: We conclude that [177Lu]Lu-FAP8-PEG3-IP-DOTA constitutes a promising and safe RPT candidate for FAPα-targeted radionuclide therapy of solid tumors.

Listeria adhesion protein orchestrates caveolae-mediated apical junctional remodeling of epithelial barrier for Listeria monocytogenes translocation.

The cellular junctional architecture remodeling by Listeria adhesion protein-heat shock protein 60 (LAP-Hsp60) interaction for Listeria monocytogenes (Lm) passage through the epithelial barrier is incompletely understood. Here, using the gerbil model, permissive to internalin (Inl) A/B-mediated pathways like in humans, we demonstrate that Lm crosses the intestinal villi at 48 h post-infection. In contrast, the single isogenic (lap- or ΔinlA) or double (lap-ΔinlA) mutant strains show significant defects. LAP promotes Lm translocation via endocytosis of cell-cell junctional complex in enterocytes that do not display luminal E-cadherin. In comparison, InlA facilitates Lm translocation at cells displaying apical E-cadherin during cell extrusion and mucus expulsion from goblet cells. LAP hijacks caveolar endocytosis to traffic integral junctional proteins to the early and recycling endosomes. Pharmacological inhibition in a cell line and genetic knockout of caveolin-1 in mice prevents LAP-induced intestinal permeability, junctional endocytosis, and Lm translocation. Furthermore, LAP-Hsp60-dependent tight junction remodeling is also necessary for InlA access to E-cadherin for Lm intestinal barrier crossing in InlA-permissive hosts. IMPORTANCE: Listeria monocytogenes (Lm) is a foodborne pathogen with high mortality (20%-30%) and hospitalization rates (94%), particularly affecting vulnerable groups such as pregnant women, fetuses, newborns, seniors, and immunocompromised individuals. Invasive listeriosis involves Lm's internalin (InlA) protein binding to E-cadherin to breach the intestinal barrier. However, non-functional InlA variants have been identified in Lm isolates, suggesting InlA-independent pathways for translocation. Our study reveals that Listeria adhesion protein (LAP) and InlA cooperatively assist Lm entry into the gut lamina propria in a gerbil model, mimicking human listeriosis in early infection stages. LAP triggers caveolin-1-mediated endocytosis of critical junctional proteins, transporting them to early and recycling endosomes, facilitating Lm passage through enterocytes. Furthermore, LAP-Hsp60-mediated junctional protein endocytosis precedes InlA's interaction with basolateral E-cadherin, emphasizing LAP and InlA's cooperation in enhancing Lm intestinal translocation. This understanding is vital in combating the severe consequences of Lm infection, including sepsis, meningitis, encephalitis, and brain abscess.

A chimeric vaccine derived from Australian genotype IV Japanese encephalitis virus protects mice from lethal challenge.

In 2022, a genotype IV (GIV) strain of Japanese encephalitis virus (JEV) caused an unprecedented and widespread outbreak of disease in pigs and humans in Australia. As no veterinary vaccines against JEV are approved in Australia and all current approved human and veterinary vaccines are derived from genotype (G) III JEV strains, we used the recently described insect-specific Binjari virus (BinJV) chimeric flavivirus vaccine technology to produce a JEV GIV vaccine candidate. Herein we describe the production of a chimeric virus displaying the structural prM and E proteins of a JEV GIV isolate obtained from a stillborn piglet (JEVNSW/22) in the genomic backbone of BinJV (BinJ/JEVNSW/22-prME). BinJ/JEVNSW/22-prME was shown to be antigenically indistinguishable from the JEVNSW/22 parental virus by KD analysis and a panel of JEV-reactive monoclonal antibodies in ELISA. BinJ/JEVNSW/22-prME replicated efficiently in C6/36 cells, reaching titres of >107 infectious units/mL - an important attribute for vaccine manufacture. As expected, BinJ/JEVNSW/22-prME failed to replicate in a variety of vertebrate cells lines. When used to immunise mice, the vaccine induced a potent virus neutralising response against JEVNSW/22 and to GII and GIII JEV strains. The BinJ/JEVNSW/22-prME vaccine provided complete protection against lethal challenge with JEVNSW/22, whilst also providing partial protection against viraemia and disease for the related Murray Valley encephalitis virus. Our results demonstrate that BinJ/JEVNSW/22-prME is a promising vaccine candidate against JEV.

Impact of Rehydration Following Systemic Dehydration on Vocal Fold Gene Expression.

OBJECTIVE: Biological data on the beneficial effects of vocal fold rehydration are lacking. This study aimed to examine the effects of acute systemic dehydration on vocal fold gene expression and determine whether rehydration would reverse these changes. METHODS: Male New Zealand White rabbits (N = 24, n = 8/group) provided the animal model. Systemic dehydration was induced by 5 days of water volume restriction. Rehydration was provided by ad-lib water for 3 days following dehydration. Euhydrated rabbits were used as the control group. Vocal fold tissue was dissected. Seventeen genes were selected based on physiological function and role in supporting vocal fold structure, oxidative stress, hemodynamics, and extracellular matrix turnover. Relative gene expression was assessed by RT-qPCR. RESULTS: Rehydration following systemic dehydration can modulate gene expression, with expression patterns suggesting that rehydration reverses dehydration-induced changes in over half of the tested genes. CLIC5 (chloride intracellular channel 5) and EFEMP1 (EGF containing fibulin extracellular matrix protein 1) genes were significantly upregulated in the dehydration group compared with the euhydrated control. A1BG (alpha-1B-glycoprotein) and IL1RAP (interleukin 1 receptor accessory protein) were downregulated by rehydration compared with the dehydration group. CONCLUSION: This study provides molecular evidence for a transcriptional response to rehydration following acute systemic dehydration in the vocal folds. These data are the first to study gene expression following realistic dehydration and rehydration paradigms and provide biological data to support clinical recommendations to increase water intake after acute dehydration. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3499-3505, 2023.

Hydration State and Hyaluronidase Treatment Significantly Affect Porcine Vocal Fold Biomechanics.

OBJECTIVES: The understanding of vocal fold hydration state, including dehydrated, euhydrated, rehydrated tissue, and how hydration affects vocal fold biomechanical properties is still evolving. Although clinical observations support the benefits of increasing vocal fold hydration after dehydrating events, more mechanistic information on the effects of vocal fold dehydration and the beneficial effects of rehydration are needed. Alterations to hyaluronic acid (HA), an important component of the vocal fold extracellular matrix, are likely to influence the biomechanical properties of vocal folds. In this study, we investigated the influence of hydration state and HA on vocal fold tissue stiffness via biomechanical testing. STUDY DESIGN: Prospective, ex vivo study design. METHODS: Fresh porcine vocal folds (N = 18) were examined following sequential immersion in hypertonic (dehydration) and isotonic solutions (rehydration). In a separate experiment, vocal folds were incubated in hyaluronidase (Hyal) to remove HA. Control tissues were not exposed to any challenges. A custom micromechanical system with a microforce sensing probe was used to measure the force-displacement response. Optical strain was calculated, and ultrasound imaging was used to measure tissue cross-sectional area to obtain stress-strain curves. RESULTS: Significant increases (P ≤ 0.05) were found in tangent moduli between dehydrated and rehydrated vocal folds at strains of ε = 0.15. The tangent moduli of Hyal-digested tissues significantly increased at both ε = 0.15 and 0.3 (P ≤ 0.05). CONCLUSION: Vocal fold dehydration increased tissue stiffness and rehydration reduced the stiffness. Loss of HA increased vocal fold stiffness, suggesting a potential mechanical role for HA in euhydrated vocal folds.

Comparative proteomic changes in rabbit vocal folds undergoing systemic dehydration and systemic rehydration.

BACKGROUND: A considerable body of clinical evidence suggests that systemic dehydration can negatively affect voice production, leading to the common recommendation to rehydrate. Evidence for the corrective benefits of rehydration, however, is limited with mixed conclusions, and biological data on the underlying tissue changes with rehydration is lacking. In this study, we used a rabbit model (n = 24) of acute (5 days) water restriction-induced systemic dehydration with subsequent rehydration (3 days) to explore the protein-level changes underlying the molecular transition from euhydration to dehydration and following rehydration using LC-MS/MS protein quantification in the vocal folds. We show that 5-day water restriction led to an average 4.3% decrease in body weight with relative increases in anion gap, Cl-, creatinine, Na+, and relative decreases in BUN, iCa2+, K+, and tCO2 compared to control (euhydrated) animals. A total of 309 differentially regulated (p < 0.05) proteins were identified between the Control and Dehydration groups. We observed a noteworthy similarity between the Dehydration and Rehydration groups, both well differentiated from the Control group, highlighting the distinct timelines of resolution of the clinical symptoms of systemic dehydration and the underlying molecular changes. SIGNIFICANCE: Voice disorders are a ubiquitous problem with considerable economic and psychological impact. Maintenance of proper hydration is commonly prescribed as a general vocal hygiene practice. There is evidence that dehydration negatively impacts phonation, but our understanding of the state of vocal folds in the context of systemic dehydration are limited, particular from a molecular perspective. Further, ours is a novel molecular study of the short-term impact of rehydration on the tissue. Given the relatively minimal difference in vocal fold proteomic profiles between the Dehydration and Rehydration groups, our data demonstrate a complex physiological response to acute systemic dehydration, and highlight the importance of considering persistent underlying molecular pathology despite the rapid resolution of clinical measures. This study sets a foundation for future research to confirm the nature of potential beneficial outcomes of clinical recommendations related to hydration.