Preventive and therapeutic effects of a super-multivalent sialylated filamentous bacteriophage against the influenza virus.

The resurgence of influenza viruses as a significant global threat emphasizes the urgent need for innovative antiviral strategies beyond existing treatments. Here, we present the development and evaluation of a novel super-multivalent sialyllactosylated filamentous phage, termed t-6SLPhage, as a potent entry blocker for influenza A viruses. Structural variations in sialyllactosyl ligands, including linkage type, valency, net charge, and spacer length, were systematically explored to identify optimal binding characteristics against target hemagglutinins and influenza viruses. The selected SLPhage equipped with optimal ligands, exhibited exceptional inhibitory potency in in vitro infection inhibition assays. Furthermore, in vivo studies demonstrated its efficacy as both a preventive and therapeutic intervention, even when administered post-exposure at 2 days post-infection, under 4 lethal dose 50% conditions. Remarkably, co-administration with oseltamivir revealed a synergistic effect, suggesting potential combination therapies to enhance efficacy and mitigate resistance. Our findings highlight the efficacy and safety of sialylated filamentous bacteriophages as promising influenza inhibitors. Moreover, the versatility of M13 phages for surface modifications offers avenues for further engineering to enhance therapeutic and preventive performance.

Antibiotic-eluting scaffolds with responsive dual-release kinetics facilitate bone healing and eliminate S. aureus infection.

Osteomyelitis (OM) is a progressive, inflammatory infection of bone caused predominately by Staphylococcus aureus. Herein, we engineered an antibiotic-eluting collagen-hydroxyapatite scaffold capable of eliminating infection and facilitating bone healing. An iterative freeze-drying and chemical crosslinking approach was leveraged to modify antibiotic release kinetics, resulting in a layered dual-release system whereby an initial rapid release of antibiotic to clear infection was followed by a sustained controlled release to prevent reoccurrence of infection. We observed that the presence of microbial collagenase accelerated antibiotic release from the crosslinked layer of the scaffold, indicating that the material is responsive to microbial activity. As exemplar drugs, vancomycin and gentamicin-eluting scaffolds were demonstrated to be bactericidal, and supported osteogenesis in vitro. In a pilot murine model of OM, vancomycin-eluting scaffolds were observed to reduce S. aureus infection within the tibia. Finally, in a rabbit model of chronic OM, gentamicin-eluting scaffolds both facilitated radial bone defect healing and eliminated S. aureus infection. These results show that antibiotic-eluting collagen-hydroxyapatite scaffolds are a one-stage therapy for OM, which when implanted into infected bone defects simultaneously eradicate infection and facilitate bone tissue healing.

Two-Photon Microscopy to Measure Calcium Signaling in the Living Brain.

Two-photon microscopy enables imaging of calcium signaling at cellular or subcellular resolution up to hundreds of microns deep in the living brain. Changes in the brightness of fluorescent calcium indicators provide a readout of calcium levels over time, affording information about neuronal activity and/or calcium-dependent subcellular signaling. Here, we describe a protocol for repeated two-photon imaging of calcium signals in mice expressing a genetically encoded calcium indicator that have been implanted with a chronic cranial window.

Upconversion nanoparticles-CuMnO2 nanoassemblies for NIR-excited imaging of reactive oxygen species in vivo.

Here, we designed a ratiometric luminescent nanoprobe based on lanthanide-doped upconversion nanoparticles-CuMnO2 nanoassemblies for rapid and sensitive detection of reactive oxygen species (ROS) levels in living cells and mouse. CuMnO2 nanosheets exhibit a wide absorption range of 300-700 nm, overlapping with the visible-light emission of upconversion nanoparticles (UCNPs), resulting in a significant upconversion luminescence quenching. In an acidic environment, H2O2 can promote the redox reaction of CuMnO2, leading to its dissociation from the surface of UCNPs and the restoration of upconversion luminescence. The variation in luminescence intensity ratio (UCL475/UCL450) were monitored to detect ROS levels. The H2O2 nanoprobe exhibited a linear response in the range of 0.314-10 µM with a detection limit of 11.3 nM. The biological tests proved the excellent biocompatibility and low toxicity of obtained UCNPs-CuMnO2 nanoassemblies. This ratiometric luminescent nanoprobe was successfully applied for the detection of exogenous and endogenous ROS in live cells as well as in vivo ROS quantitation. The dual transition metal ions endow this probe efficient catalytic decomposition capabilities, and this sensing strategy broadens the application of UCNPs-based nanomaterials in the field of biological analysis and diagnosis.

PMCA to demonstrate prions inactivation method efficacy on reusable medical devices: a relevant alternative to animal bioassays.

Validation of prion inactivation processes for medical devices relies on in vivo experimental protocols. However, bioassays are costly, long (one to two years) and ethically disputable. Additionally, results obtained with one prion strain, for example 263K (hamster-adapted strain originating from sheep scrapie), cannot be easily extrapolated to relevant human prion strains, further questioning the utility of bioassays. Over the past two decades, cell-free prion amplification assays have emerged as potential alternatives to bioassays. Rather than measuring prion infectivity, they quantify prion seeding activity, i.e. the capacity to convert the normal prion protein into the disease-associated isoform. The results obtained by an optimized cell-free assay termed miniaturized-bead protein misfolding cyclic amplification (mb-PMCA), with four processes using three different prion strains, 263K and two human prions derived from variant and sporadic Creutzfeldt-Jakob diseases, were compared to published bioassays using the same three strains and processes, when available. Tests performed on reference processes (steam, sodium hydroxide, sodium hypochlorite) and low temperature H2O2 sterilization process (STERRAD NXTM Advanced cycle), showed perfect alignment between mb-PMCA and available bioassays. STERRAD NXTM Advanced cycle was efficacious on all three prion strains. These data confirm that PMCA and in particular mb-PMCA is a relevant alternative to animal bioassays for assessment of prion inactivation processes and the interest of some low temperature H2O2 sterilization cycles.

A novel peptide-based tau aggregation inhibitor as a potential therapeutic for Alzheimer's disease and other tauopathies.

INTRODUCTION: As aggregation underpins Tau toxicity, aggregation inhibitor peptides may have disease-modifying potential. They are therefore currently being designed and target either the 306VQIVYK311 aggregation-promoting hotspot found in all Tau isoforms or the 275VQIINK280 aggregation-promoting hotspot found in 4R isoforms. However, for any Tau aggregation inhibitor to potentially be clinically relevant for other tauopathies, it should target both hotspots to suppress aggregation of Tau isoforms, be stable, cross the blood-brain barrier, and rescue aggregation-dependent Tau phenotypes in vivo. METHODS: We developed a retro-inverso, stable D-amino peptide, RI-AG03 [Ac-rrrrrrrrGpkyk(ac)iqvGr-NH2], based on the 306VQIVYK311 hotspots which exhibit these disease-relevant attributes. RESULTS: Unlike other aggregation inhibitors, RI-AG03 effectively suppresses aggregation of multiple Tau species containing both hotspots in vitro and in vivo, is non-toxic, and suppresses aggregation-dependent neurodegenerative and behavioral phenotypes. DISCUSSION: RI-AG03 therefore meets many clinically relevant requirements for an anti-aggregation Tau therapeutic and should be explored further for its disease-modifying potential for Tauopathies. HIGHLIGHTS: Our manuscript describes the development of a novel peptide inhibitor of Tau aggregation, a retro-inverso, stable D-amino peptide called RI-AG03 that displays many clinically relevant attributes. We show its efficacy in preventing Tau aggregation in both in vitro and in vivo experimental models while being non-toxic to cells. RI-AG03 also rescues a biosensor cell line that stably expresses Tau repeat domains with the P301S mutation fused to Cer/Clo and rescues aggregation-dependent phenotypes in vivo, suppressing neurodegeneration and extending lifespan. Collectively our data describe several properties and attributes of RI-AG03 that make it a promising disease-modifying candidate to explore for reducing pathogenic Tau aggregation in Tauopathies such as Alzheimer's disease. Given the real interest in reducing Tau aggregation and the potential clinical benefit of using such agents in clinical practice, RI-AG03 should be investigated further for the treatment of Tauopathies after validation in mammalian models. Tau aggregation inhibitors are the obvious first choice as Tau-based therapies as much of Tau-mediated toxicity is aggregation dependent. Indeed, there are many research efforts focusing on this therapeutic strategy with aggregation inhibitors being designed against one of the two aggregation-promoting hotspots of the Tau protein. To our knowledge, RI-AG03 is the only peptide aggregation inhibitor that inhibits aggregation of Tau by targeting both aggregation-promoting hotspot motifs simultaneously. As such, we believe that our study will have a significant impact on drug discovery efforts in this arena.

"Yin-Yang philosophy" for the design of anticancer drug delivery nanoparticles.

Understanding the in vivo transport process provides guidelines for designing ideal nanoparticles (NPs) with higher efficacy and fewer off-target effects. Many factors, such as particle size, morphology, surface potential, structural stability, and etc., may influence the delivering process of NPs due to the existence of various physiological barriers within the body. Herein, we summarise the distinct influences of NP physicochemical properties on the four consecutive in vivo transport steps: (1) navigating with bloodstream within blood vessels, (2) transport across vasculature walls into tumour tissues, (3) intratumoural transport through the interstitial space, and (4) cellular uptake & intracellular delivery by cancerous cells. We found that the philosophy behind the current consensus for NP design has certain similarities to the "Yin-Yang" theory in traditional Chinese culture. Almost all physicochemical properties, regardless of big or small sizes, long or short length, positive or negative zeta potentials, are double-edged swords. The balance of potential benefits and side effects, drug selectivity and accessibility should be fully considered when optimising particle design, similar to the "Yin-Yang harmony". This paper presents a comprehensive review of the advancements in NPs research, focusing on their distinct features in tumour targeting, drug delivery, and cell uptake. Additionally, it deliberates on future developmental trends and potential obstacles, thereby aiming to uncover the ways these characteristics influence the NPs' biological activity and provide theoretical guidance for the targeted delivery of NPs.

Elucidation of the mechanisms of fluconazole resistance and repurposing treatment options against urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt.

BACKGROUND: The incidence of fungal urinary tract infections (UTIs) has dramatically increased in the past decades, with Candida arising as the predominant etiological agent. Managing these infections poses a serious challenge to clinicians, especially with the emergence of fluconazole-resistant (FLC-R) Candida species. In this study, we aimed to determine the mechanisms of fluconazole resistance in urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt, assess the correlation between fluconazole resistance and virulence, and explore potential treatment options for UTIs caused by FLC-R Candida strains. RESULTS: Fluconazole susceptibility testing of 34 urinary Candida isolates indicated that 76.5% were FLC-R, with a higher prevalence of resistance recorded in non-albicans Candida spp. (88.9%) than in Candida albicans (62.5%). The calculated Spearman's correlation coefficients implied significant positive correlations between fluconazole minimum inhibitory concentrations and both biofilm formation and phospholipase production. Real-time PCR results revealed that most FLC-R isolates (60%) significantly overexpressed at least one efflux pump gene, while 42.3% significantly upregulated the ERG11 gene. The most prevalent mutation detected upon ERG11 sequencing was G464S, which is conclusively linked to fluconazole resistance. The five repurposed agents: amikacin, colistin, dexamethasone, ketorolac, and sulfamethoxazole demonstrated variable fluconazole-sensitizing activities in vitro, with amikacin, dexamethasone, and colistin being the most effective. However, the fluconazole/colistin combination produced a notable reduction (49.1%) in bladder bioburden, a 50% decrease in the inflammatory response, and tripled the median survival span relative to the untreated murine models. CONCLUSIONS: The fluconazole/colistin combination offers a promising treatment option for UTIs caused by FLC-R Candida, providing an alternative to the high-cost, tedious process of novel antifungal drug discovery in the battle against antifungal resistance.

A critical review of bioanalytical and clinical applications of solid phase microextraction.

Studying the functions, mechanisms, and effects of drugs and other exogenous compounds on biological systems, together with investigations performed to understand biosystems better, comprises one of the most fascinating areas of research. Although classical sample preparation techniques are dominantly used to infer the relevant information from the investigated system, they fail to meet various imperative requirements, such as being environmentally friendly, applicable in-vivo, and compatible with online analysis. As a chameleon in the analytical toolbox, solid phase microextraction (SPME) is one of the best tools available for studying biological systems in unconventional ways. In this review, SPME is spotlighted, and its capability for bioanalytical applications, including drug analysis, untargeted and targeted metabolomics, in-vivo and clinical studies, is scrutinized based on studies reported in the past five years. In addition, novel extractive phases and instrumental coupling strategies developed to serve bioanalytical research are discussed to give the perspective for state-of-the-art and future developments. The literature assessment showed that SPME could act as a critical tool to investigate in-vivo biological systems and provide information about the elusive portion of the metabolome. Moreover, recently introduced miniaturized SPME probes further improved the low-invasive nature of the sampling and enabled sampling even from a single cell. The coupling of SPME directly to mass spectrometry significantly reduced the total analytical workflow and became one of the promising tools suitable for fast diagnostic purposes and drug analysis. The numerous applications and advancements reported in bioanalysis using SPME show that it will continue to be an indispensable technique in the future.

Discovering the most impactful treatments for aluminum phosphide cardiotoxicity gleaned from systematic review of animal studies.

INTRODUCTION: Aluminum phosphide (AlP) is a chemical compound that can cause death in some countries. AlP inhibits the functioning of cytochrome C oxidase in the mitochondria of cardiomyocytes, leading to toxicity. Oxidative stress and ROS production, as well as inflammatory signaling, mediate the mechanisms of AlP-related toxicity in the poisoned patient. Unfortunately, there are no approved medicines available to treat AlP poisoning yet. To address this issue, researchers have explored various interventions to reduce the toxicity associated with AlP tablets. METHODS: We systematically searched relevant databases for English articles published between 2013 and 2024. RESULTS: The evaluated treatments included correcting oxidative stress parameters, enhancing exogenous antioxidant capacity, modifying electrocardiographic abnormalities, and improving heart contraction strength. Our evaluation indicated that compounds like Triiodothyronine, Vasopressin and milrinone, Iron sucrose, Acetyl-l-carnitine, Melatonin, Fresh red blood cell transfusion, Minocycline, Moringa oleifera extract, Dihydroxyacetone, Selegiline, Nanocurcumin, Levosimendan, Exenatide, Taurine, Cannabidiol and Edaravone are effective in lessening AlP-induced cardiotoxicity. CONCLUSION: Based on the present study's findings and the evaluation of clinical studies, dihydroxyacetone, fresh red blood cell infusion, Oil-based disinfection, and gastric lavage have the most potential to save patients' lives and treat acute aluminum phosphide. However, there is a need for more research in this regard.

Antiurolithic Activity of Zaleya Pentandra (L.) C Jeffrey in Ethylene glycol-induced Calcium Oxalate Crystal Rat Model; A Scientific Validation of Traditional Use for Kidney Stone Prevention.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional herbal remedies have been used for treating nephrolithiasis, but the relevant scientific evidence is limited. Zaleya pentandra (L.) C. Jeffrey is traditionally used for the prevention of kidney stones in various cultures. However, its efficacy has not been scientifically studied. AIM OF THE STUDY: This study aimed to investigate the antiurolithic activity of Zaleya pentandra, and validate its traditional used for preventing kidney stones. MATERIALS AND METHODS: The crude ethanolic extract of Z. pentandra (Zp.Crd) was evaluated through in vitro and in vivo studies. In vitro experiments assessed its impact on crystal count and morphology in metastable calcium oxalate solutions. In vivo studies involved diuretic and ethylene glycol-induced calcium oxalate crystal formation in male Wistar rats. RESULTS: Zp.Crd transforms calcium oxalate crystals from harmful calcium oxalate monohydrate (COM) to calcium oxalate dihydrate (COD). In vivo, Zp.Crd exhibited dose-dependent (30-300 mg/kg) diuretic activity in rats by significantly increasing urinary sodium (Na+) and potassium (K+) excretion, similar to the standard diuretic hydrochlorothiazide (HCT). In the urolithiasis model, Zp.Crd exhibited dose-dependent antiurolithic effects by reducing kidney crystals and significantly altering lithogenic factors induced by ethylene glycol, including crystalluria, oxaluria, calcium, creatinine, and urea, in the urine and serum of treated rats. Zp.Crd also exhibited antioxidant effects, effectively combating oxidative lithogenic stress in rats. CONCLUSION: Zp.Crd has been shown to demonstrate antiurolithic activity against CaOx stones through CaOx crystal inhibition, diuretic activity, antioxidant properties, hypocalciuric effects, and hypercitrauric actions. The findings underscore Zp.Crd's potential as a viable alternative or supplemental therapy to current urolithiasis treatments, paving the door for further clinical trials and its inclusion into modern medical practices.

Antifungal Activity of Novel Isoindoline-2-yl Putrescines as Potential Autophagy-Activated Fungicide.

The exacerbation of plant fungal diseases necessitates the development of new fungicides to prevent outbreaks. In this study, five novel isoindoline-2-yl putrescines (ISPs) were synthesized, and their synthetic procedures and gram-scale preparation were explored. When tested at 50 µg mL-1, ISPs did not significantly inhibit mycelial growth on agar plates. However, at 100 µg mL-1, they demonstrated remarkable in vivo efficacy in mitigating Botrytis cinerea infection, especially ISP3 showed curative and protective activities of 91.9% and 92.6%, respectively. Moreover, ISP3 also effectively halted lesion expansion of gray mold, Sclerotic rot, and Fusarium scabs, while inducing excessive malformed top mycelial branches of B. cinerea and Sclerotinia sclerotiorum, suppressing sclerotia formation in S. sclerotiorum, and triggering autophagic vacuolization with numerous autophagosomes in the mycelia of these fungi. Molecular docking revealed that ISP3 effectively bound to the active site of BcAtg3, forming hydrogen bonds with Ser279, Gly343, Asp370, and Asp13, along with establishing a stable salt bridge with Asp13. Furthermore, ISP3 possessed favorable ADMET properties. These findings highlight ISP3 as a promising antifungal candidate through autophagy activation.

In vivo Multi-perspective 3D + t Ultrasound Imaging and Motion Estimation of Abdominal Aortic Aneurysms.

Time-resolved three-dimensional ultrasound (3D + t US) is a promising imaging modality for monitoring abdominal aortic aneurysms (AAAs), providing their 3D geometry and motion. The lateral contrast of US is poor, a well-documented drawback which multi-perspective (MP) imaging could resolve. This study aims to show the feasibility of in vivo multi-perspective 3D + t ultrasound imaging of AAAs for improving the image contrast and displacement accuracy. To achieve this, single-perspective (SP) aortic ultrasound images from three different angles were spatiotemporally registered and fused, and the displacements were compounded. The fused MP had a significantly higher wall-lumen contrast than the SP images, for both patients and volunteers (P < .001). MP radial displacements patterns are smoother than SP patterns in 67% of volunteers and 92% of patients. The MP images from three angles have a decreased tracking error (P < .001 for all participants), and an improved SNRe compared to two out of three SP images (P < .05). This study has shown the added value of MP 3D + t US, improving both image contrast and displacement accuracy in AAA imaging. This is a step toward using multiple or large transducers in the clinic to capture the 3D geometry and strain more accurately, for patient-specific characterization of AAAs.

Relieving postherpetic neuralgia pain via gabapentin-loaded bigels as an auspicious topical drug delivery system.

BACKGROUND: Over the past decades, a substantial portion of the population worldwide has been infected with varicella zoster and most cases developed shingles. Unfortunately, shingles is usually accompanied by postherpetic neuralgia, which may persist for months to years after the resolution of the viral infection. OBJECTIVES: Gabapentin is an orally gamma-aminobutyric acid analogue approved by the Food and Drug Administration to manage shingles postherpetic neuralgia. However, gabapentin shows nonlinear pharmacokinetics, with variable absorption and bioavailability along with its short half-life and long side effects that may include dizziness and somnolence, which calls for an appropriate topical dosage form. Bigels are unique semisolid dosage forms with boosted penetrability and satisfactory hydrophilic texture. METHODS: The current work pointed to formulating gabapentin-loaded bigels for the treatment of postherpetic neuralgia, where the analysis and optimization of design were performed via Design-Expert®. RESULTS AND CONCLUSIONS: The selected bigel (F5), incorporating 400 mg Span 60, 1000 mg Tween 80, and 1000 mg Transcutol, displayed spherical nanosized particles with acceptable viscosity and spreadability. Subsequent topical application of the selected bigel on the skin of Wistar rats, F5, demonstrated a boosted accumulation of gabapentin in the skin similar to PLO gel but superior to the drug solution. Furthermore, a histopathological study demonstrated the biosafety of the selected bigel when applied topically. Accordingly, gabapentin-loaded bigel would be considered a potentially topical dosage form for the delivery of gabapentin for the management of postherpetic neuralgia.

Double-layer optical fiber interferometer with bio-layer-modified reflector for label-free biosensing of inflammatory proteins.

This work discusses label-free biosensing application of a double-layer optical fiber interferometer where the second layer tailors the reflection conditions at the external plain and supports changes in reflected optical spectrum when a bio-layer binds to it. The double-layer nanostructure consists of precisely tailored thin films, i.e., titanium (TiO2) and hafnium oxides (HfO2) deposited on single-mode fiber end-face by magnetron sputtering. It has been shown numerically and experimentally that the approach besides well spectrally defined interference pattern distinguishes refractive index (RI) changes taking place in a volume and on the sensor surface. These are of interest when label-free biosensing applications are considered. The case of myeloperoxidase (MPO) detection-a protein, which concentration rises during inflammation-is reported as an example of application. The response of the sensor to MPO in a concentration range of 1 × 10-11-5 × 10-6 g/mL was tested. An increase in the MPO concentration was followed by a redshift of the interference pattern and a decrease in reflected power. The negative control performed using ferritin proved specificity of the sensor. The results reported in this work indicate capability of the approach for diagnostic label-free biosensing, possibly also at in vivo conditions.

Biocompatibility analysis of titanium bone wedges coated by antibacterial ceramic-polymer layer.

This paper presents the surface treatment results of titanium, veterinary bone wedges. The functional coating is composed of a porous oxide layer (formed by a plasma electrolytic oxidation process) and a polymer poly(sebacic anhydride) (PSBA) layer loaded with amoxicillin (formed by dip coatings). The coatings were porous and composed of Ca (4.16%-6.54%) and P (7.64%-9.89% determined by scanning electron microscopy with EDX) in the upper part of the implant. The titanium bone wedges were hydrophilic (54° water contact angle) and rough (surface area (Sa):1.16 µm) The surface tension determined using diiodomethane was 68.6 ± 2.0° for the anodized implant and was similar for hybrid coatings: 60.7 ± 2.2°. 12.87 ± 0.91 µg/mL of amoxicillin was released from the implants during the first 30 min after immersion in the phosphate-buffered saline (PBS) solution. This concentration was enough to inhibit the Staphylococcus aureus ATCC 25923, and Staphylococcus epidermidis ATCC12228 growth. The obtained inhibition zones were between 27.3 ± 2.1 mm-30.7 ± 0.6 mm when implant extract after 1 h or 4 h immersion in PBS was collected. Various implant biocompatibility analyses were performed under in vivo conditions, including pyrogen test (3 rabbits), intracutaneous reactivity (3 rabbits, 5 places by side), acute systemic toxicity (20 house mice), and local lymph node assay (LLNA) (20 house mice). The extracts from implants were collected in polar and non-polar solutions, and the tests were conducted according to ISO 10993 standards. The results from the in vivo tests showed, that the implant's extracts are not toxic (mass body change below 5%), not sensitizing (SI < 1.6), and do not show the pyrogen effect (changes in the temperature 0.15ºC). The biocompatibility tests were performed in a certificated laboratory with a good laboratory practice certificate after all the necessary permissions.

Assessment of the risks associated with the use of in vivo versus in vitro potency tests for vaccines.

Animal (in vivo) potency tests have been utilized for over a century in support of vaccine development and for quality testing. This is a legacy of the best science at the time of their introduction. Advances in knowledge and technology, however, have provided opportunities to utilize more sensitive assays during development and replace legacy animal tests with in vitro alternatives. This coupled with initiatives such as replacement, reduction, and refinement (the 3-R's) and quality by design (QbD) have brought industry and regulators together in the introduction of advanced vaccine control strategies. This article examines historical and current uses of animals in vaccines technical development and control, and their replacement with in vitro alternatives from a risk point of view. An overarching risk is that a vaccine tested with an alternative potency assay fails to protect its target recipient. This can be addressed from the perspective of the assay's association with the vaccine mechanism of action, and the rules used to introduce the vaccine into the patient population (e.g., specifications). Commonly understood concepts such as analytical precision play a role in risk evaluation based on its impact on the sensitivity of a test to detect meaningful product changes caused by variations in manufacture or over a vaccine's shelf life. This should be considered when evaluating solutions such as the reduction of multi-concentration (or dilution) in vivo assays to a single concentration test. While the use of animals in vaccine development will not go away all together, the paradigm must shift from in vivo tests to in vivo models. To help ensure success, principles and practices related to introduction of in vitro alternatives require global collaboration among industry, regulators, pharmacopeias, and supporting organizations.

A comparative evaluation of the composition and antioxidant activity of free and bound polyphenols in sugarcane tips.

The sugarcane tip is abundant in phenolic compounds. Previous studies have concentrated on the effects of free polyphenols, while bound polyphenols were overlooked. In this study, the content of bound polyphenols (SPB) (31.9 ± 0.9 mg GAE/g DW) was significantly higher than free polyphenols (SPF) (3.4 ± 0.1 mg GAE/g DW). A total of 44 free and 31 bound phenolics were identified by the UPLC-EIS-QTOF-MS/MS. Moreover, the antioxidant activity of SPB was more pronounced, as evidenced by its higher ABTS+ and DPPH scavenging rates than SPF, which was attributed to the higher tannin content. Furthermore, at all tested concentrations (100 and 200 µg/mL), SPB significantly enhanced the survival and antioxidant enzyme activity of Caenorhabditis elegans (C. elegans), while concurrently reducing ROS levels. High concentrations of SPB even exhibited antioxidant activity comparable to Vitamin C (Vc). The collective findings strongly indicate that SPB holds great potential as an effective antioxidant.

Effect of mutations in GDNV motif of viral hemorrhagic septicemia virus (VHSV) L protein on polymerase activity, viral growth, and in vivo virulence.

Most Mononegavirales viruses have a GDNQ motif within the L protein, whereas Novirhabdovirus species feature a GDNV motif. This study examined the function of the GDNV motif within the L protein of viral hemorrhagic septicemia virus (VHSV) by modifying its amino acid composition. Substituting the aspartic acid (D) with valine (V) completely abolished polymerase activity in a minigenome assay. Replacing GDNV with GDNQ showed no significant difference in luciferase activity. Further characterization using reverse genetically engineered recombinant viruses revealed that rVHSV-LGDNQ exhibited an accelerated replication rate and higher virus titer in EPC cells than rVHSV-wild. Olive flounder infected with rVHSV-LGDNQ experienced higher early-stage mortality but lower overall mortality than those infected with rVHSV-wild. These findings suggest that while the GDNQ motif may positively influence VHSV replication speed, it may not confer an overall advantage for the ultimate viral pathogenicity.

Recording γ-secretase activity in living mouse brains.

γ-Secretase plays a pivotal role in the central nervous system. Our recent development of genetically encoded Förster resonance energy transfer (FRET)-based biosensors has enabled the spatiotemporal recording of γ-secretase activity on a cell-by-cell basis in live neurons in culture. Nevertheless, how γ-secretase activity is regulated in vivo remains unclear. Here, we employ the near-infrared (NIR) C99 720-670 biosensor and NIR confocal microscopy to quantitatively record γ-secretase activity in individual neurons in living mouse brains. Intriguingly, we uncovered that γ-secretase activity may influence the activity of γ-secretase in neighboring neurons, suggesting a potential 'cell non-autonomous' regulation of γ-secretase in mouse brains. Given that γ-secretase plays critical roles in important biological events and various diseases, our new assay in vivo would become a new platform that enables dissecting the essential roles of γ-secretase in normal health and diseases.