Preclinical Settings Research Articles

A Trapping-Micro-LC-FAIMS/dCV-MS Strategy for Ultrasensitive and Robust Targeted Quantification of Protein Drugs and Biomarkers.

The sensitivity of LC-MS in quantifying target proteins in plasma/tissues is significantly hindered by coeluted matrix interferences. While antibody-based immuno-enrichment effectively reduces interferences, developing and optimizing antibodies are often time-consuming and costly. Here, by leveraging the orthogonal separation capability of Field Asymmetric Ion Mobility Spectrometry (FAIMS), we developed a FAIMS/differential-compensation-voltage (FAIMS/dCV) method for antibody-free, robust, and ultrasensitive quantification of target proteins directly from plasma/tissue digests. By comparing the intensity-CV profiles of the target vs coeluted endogenous interferences, the FAIMS/dCV approach identifies the optimal CV for quantification of each target protein, thus maximizing the signal-to-noise ratio (S/N). Compared to quantification without FAIMS, this technique dramatically reduces endogenous interferences, showing a median improvement of the S/N by 14.8-fold for the quantification of 17 representative protein drugs and biomarkers in plasma or tissues and a 5.2-fold median increase in S/N over conventional FAIMS approach, which uses the peak CV of each target. We also discovered that the established CV parameters remain consistent over months and are matrix-independent, affirming the robustness of the developed FAIMS/dCV method and the transferability of the method across matrices. The developed method was successfully demonstrated in three applications: the quantification of monoclonal antibodies with subng/mL LOQ in plasma, an investigation of the time courses of evolocumab and its target PCSK9 in a preclinical setting, and a clinical investigation of low abundance obesity-related biomarkers. This innovative and easy-to-use method has extensive potential in clinical and pharmaceutical research, particularly where sensitive and high-throughput quantification of protein drugs and biomarkers is required.

Potential Targeting Mechanisms for Bone-Directed Therapies.

Bone development is characterized by complex regulation mechanisms, including signal transduction and transcription factor-related pathways, glycobiological processes, cellular interactions, transportation mechanisms, and, importantly, chemical formation resulting from hydroxyapatite. Any abnormal regulation in the bone development processes causes skeletal system-related problems. To some extent, the avascularity of cartilage and bone makes drug delivery more challenging than that of soft tissues. Recent studies have implemented many novel bone-targeting approaches to overcome drawbacks. However, none of these strategies fully corrects skeletal dysfunction, particularly in growth plate-related ones. Although direct recombinant enzymes (e.g., Vimizim for Morquio, Cerezyme for Gaucher, Elaprase for Hunter, Mepsevii for Sly diseases) or hormone infusions (estrogen for osteoporosis and osteoarthritis), traditional gene delivery (e.g., direct infusion of viral or non-viral vectors with no modifications on capsid, envelope, or nanoparticles), and cell therapy strategies (healthy bone marrow or hematopoietic stem cell transplantation) partially improve bone lesions, novel delivery methods must be addressed regarding target specificity, less immunogenicity, and duration in circulation. In addition to improvements in bone delivery, potential regulation of bone development mechanisms involving receptor-regulated pathways has also been utilized. Targeted drug delivery using organic and inorganic compounds is a promising approach in mostly preclinical settings and future clinical translation. This review comprehensively summarizes the current bone-targeting strategies based on bone structure and remodeling concepts while emphasizing potential approaches for future bone-targeting systems.

Nanotechnology applications in breast implant manufacturing for improved durability and functionality

Breast implants are often used in restorative and cosmetic surgeries. However, the materials used for implants today could be better regarding biocompatibility and mechanical strength. It is possible to make composite biomaterials out of nanomaterials with better mechanical qualities than common implant materials like silicone elastomers and saline-filled shells. Researchers have found that adding carbon nanotubes, graphene, and hydroxyapatite nanoparticles to the shells of metal and silicone implants makes them much stronger, more flexible, and less likely to break. Researchers are also looking into polymer nanocomposites made of polycaprolactone and polylactic acid to see if they can break down better and integrate better with tissues. Changing the surface of implants at the nanoscale level can make them more biocompatible by controlling how proteins stick to the material and how cells interact with it. Animal tests with nanocoatings of polyacrylate, chitosan, and hyaluronic acid showed that they lowered the formation of capsules and inflammation. Antimicrobial nanoparticles, such as silver, zinc oxide, and antibiotics, are attached to the surfaces of implants to protect against infections and release drugs locally. Nanocontrast agents are used in high-resolution MRI and ultrasound images of implant shell integrity to get new imaging and diagnosis tools. By tracking biomarkers, nanostructured sensors could be used to find seromas, ruptures, and device failures with little to no damage. Much work has been done to show that different nanotechnologies can help breast implants in a pre-clinical setting. However, problems with regulation and standardization still need to be fixed before the implants can be used in people and made in large quantities. The process needs to be improved even more to make a lot of nanocomposite and etched surfaces. To make sure patients are safe, it is also important to do long-term biocompatibility and nanotoxicology tests. Nanotechnology has a lot of promise to change the way breast implants are made so that they look better and improve people's quality of life. This review examines nanotechnology's emerging applications for enhancing breast implants' durability and performance.

An update on novel and emerging therapeutic targets in Parkinson's disease.

Parkinson's Disease (PD) remains a significant focus of extensive research aimed at developing effective therapeutic strategies. Current treatments primarily target symptom management, with limited success in altering the course of the disease. This shortfall underscores the urgent need for novel therapeutic approaches that can modify the progression of PD.This review concentrates on emerging therapeutic targets poised to address the underlying mechanisms of PD. Highlighted novel and emerging targets include Protein Abelson, Rabphilin-3A, Colony Stimulating Factor 1-Receptor, and Apelin, each showing promising potential in preclinical and clinical settings for their ability to modulate disease progression. By examining recent advancements and outcomes from trials focusing on these targets, the review aims to elucidate their efficacy and potential as disease-modifying therapies.Furthermore, the review explores the concept of multi-target approaches, emphasizing their relevance in tackling the complex pathology of PD. By providing comprehensive insights into these novel targets and their therapeutic implications, this review aims to guide future research directions and clinical developments toward more effective treatments for PD and related neurodegenerative disorders.

Class II ferroptosis inducers are a novel therapeutic approach for t(4;14)-positive multiple myeloma

AbstractMultiple myeloma (MM) is a clonal plasma cell malignancy that is characterized by genetic heterogeneity. The cytogenetic abnormality t(4;14) strongly predicts poor outcome in patients with MM, even in the era of novel drugs. Ferroptosis is a new approach to antitumor therapy, but the relationship between ferroptosis and MM cytogenetic abnormalities remains largely unclear. In this study, we show that t(4;14)-positive but not t(4;14)-negative MM cells are susceptible to class II ferroptosis inducers (FINs) in a preclinical setting, which is dependent on the significant upregulation of the MM SET domain-containing protein (MMSET). Mechanistically, MMSET upregulates acyl-coenzyme A synthetase long-chain family member 4 transcription by binding to its promoter region, leading to increased polyunsaturated fatty acid (PUFA) levels and enhanced sensitivity of t(4;14)-positive MM cells to ferroptosis. Supplementation with PUFAs efficiently restores the susceptibility of t(4;14)-negative MM cells to ferroptosis. In addition, combining class II FIN treatment with bortezomib in t(4;14)-positive MM cells attenuates cellular glutathione and induces both apoptosis and ferroptosis levels by inhibiting the increase in solute carrier family 7 member 11, demonstrating synergistic antitumor activity in vitro and in a xenograft model. Taken together, our findings suggest that targeting ferroptosis with class II FINs is a novel and promising therapeutic approach to improve the outcome of t(4;14)-positive patients with MM.

Methylome-proteome integration after late-life voluntary exercise training reveals regulation and target information for improved skeletal muscle health.

Exercise is a potent stimulus for combatting skeletal muscle ageing. To study the effects of exercise on muscle in a preclinical setting, we developed a combined endurance-resistance training stimulus for mice called progressive weighted wheel running (PoWeR). PoWeR improves molecular, biochemical, cellular and functional characteristics of skeletal muscle and promotes aspects of partial epigenetic reprogramming when performed late in life (22-24months of age). In this investigation, we leveraged pan-mammalian DNA methylome arrays and tandem mass-spectrometry proteomics in skeletal muscle to provide detailed information on late-life PoWeR adaptations in female mice relative to age-matched sedentary controls (n=7-10 per group). Differential CpG methylation at conserved promoter sites was related to transcriptional regulation genes as well as Nr4a3, Hes1 and Hox genes after PoWeR. Using a holistic method of -omics integration called binding and expression target analysis (BETA), methylome changes were associated with upregulated proteins related to global and mitochondrial translation after PoWeR (P=0.03). Specifically, BETA implicated methylation control of ribosomal, mitoribosomal, and mitochondrial complex I protein abundance after training. DNA methylation may also influence LACTB, MIB1 and UBR4 protein induction with exercise - all are mechanistically linked to muscle health. Computational cistrome analysis predicted several transcription factors including MYC as regulators of the exercise trained methylome-proteome landscape, corroborating prior late-life PoWeR transcriptome data. Correlating the proteome to muscle mass and fatigue resistance revealed positive relationships with VPS13A and NPL levels, respectively. Our findings expose differential epigenetic and proteomic adaptations associated with translational regulation after PoWeR that could influence skeletal muscle mass and function in aged mice. KEY POINTS: Late-life combined endurance-resistance exercise training from 22-24months of age in mice is shown to improve molecular, biochemical, cellular and in vivo functional characteristics of skeletal muscle and promote aspects of partial epigenetic reprogramming and epigenetic age mitigation. Integration of DNA CpG 36k methylation arrays using conserved sites (which also contain methylation ageing clock sites) with exploratory proteomics in skeletal muscle extends our prior work and reveals coordinated and widespread regulation of ribosomal, translation initiation, mitochondrial ribosomal (mitoribosomal) and complex I proteins after combined voluntary exercise training in a sizeable cohort of female mice (n=7-10 per group and analysis). Multi-omics integration predicted epigenetic regulation of serine β-lactamase-like protein (LACTB - linked to tumour resistance in muscle), mind bomb 1 (MIB1 - linked to satellite cell and type 2 fibre maintenance) and ubiquitin protein ligase E3 component N-recognin 4 (UBR4 - linked to muscle protein quality control) after training. Computational cistrome analysis identified MYC as a regulator of the late-life training proteome, in agreement with prior transcriptional analyses. Vacuolar protein sorting 13homolog A (VPS13A) was positively correlated to muscle mass, and the glycoprotein/glycolipid associated sialylation enzyme N-acetylneuraminate pyruvate lyase (NPL) was associated to in vivo muscle fatigue resistance.

Impact of undersampling on preclinical lung T2* mapping with 3D radial UTE MRI at 7 T

Lung diseases are almost invariably heterogeneous and progressive, making it imperative to capture temporally and spatially explicit information to understand the disease initiation and progression. Imaging the lung with MRI—particularly in the preclinical setting—has historically been challenging because of relatively low lung tissue density, rapid cardiac and respiratory motion, and rapid transverse (T2*) relaxation. These limitations can largely be mitigated using ultrashort-echo-time (UTE) sequences, which are intrinsically robust to motion and avoid significant T2* decay. A significant disadvantage of common radial UTE sequences is that they require inefficient, center-out k-space sampling, resulting in long acquisition times relative to conventional Cartesian sequences. Therefore, pulmonary images acquired with radial UTE are often undersampled to reduce acquisition time. However, undersampling reduces image SNR, introduces image artifacts, and degrades true image resolution. The level of undersampling is further increased if offline gating techniques like retrospective gating are employed, because only a portion (∼40–50%) of the data is used in the final image reconstruction. Here, we explore the impact of undersampling on SNR and T2* mapping in mouse lung imaging using simulation and in-vivo data. Increased scatter in both metrics was noticeable at around 50% sampling. Parenchymal apparent SNR only decreased slightly (average decrease ∼ 1.4) with as little as 10% sampling. Apparent T2* remained similar across undersampling levels, but it became significantly increased (p < 0.05) below 80% sampling. These trends suggest that undersampling can generate quantifiable, but moderate changes in the apparent value of T2*. Moreover, these approaches to assess the impact of undersampling are straightforward to implement and can readily be expanded to assess the quantitative impact of other MR acquisition and reconstruction parameters.

Anti-Neuroinflammatory Effects of Ginkgo biloba Extract EGb 761 in LPS-Activated BV2 Microglial Cells.

Inflammatory processes in the brain can exert important neuroprotective functions. However, in neurological and psychiatric disorders, it is often detrimental due to chronic microglial over-activation and the dysregulation of cytokines and chemokines. Growing evidence indicates the emerging yet prominent pathophysiological role of neuroinflammation in the development and progression of these disorders. Despite recent advances, there is still a pressing need for effective therapies, and targeting neuroinflammation is a promising approach. Therefore, in this study, we investigated the anti-neuroinflammatory potential of a marketed and quantified proprietary herbal extract of Ginkgo biloba leaves called EGb 761 (10-500 µg/mL) in BV2 microglial cells stimulated by LPS (10 ng/mL). Our results demonstrate significant inhibition of LPS-induced expression and release of cytokines tumor necrosis factor-α (TNF-α) and Interleukin 6 (IL-6) and chemokines C-X-C motif chemokine ligand 2 (CXCL2), CXCL10, c-c motif chemokine ligand 2 (CCL2) and CCL3 in BV2 microglial cells. The observed effects are possibly mediated by the mitogen-activated protein kinases (MAPK), p38 MAPK and ERK1/2, as well as the protein kinase C (PKC) and the nuclear factor (NF)-κB signaling cascades. The findings of this in vitro study highlight the anti-inflammatory properties of EGb 761 and its therapeutic potential, making it an emerging candidate for the treatment of neuroinflammatory diseases and warranting further research in pre-clinical and clinical settings.

Comparison of the tolerability of 161Tb- and 177Lu-labeled somatostatin analogues in the preclinical setting.

[177Lu]Lu-DOTATATE is an established somatostatin receptor (SSTR) agonist for the treatment of metastasized neuroendocrine neoplasms, while the SSTR antagonist [177Lu]Lu-DOTA-LM3 has only scarcely been employed in clinics. Impressive preclinical data obtained with [161Tb]Tb-DOTA-LM3 in tumor-bearing mice indicated the potential of terbium-161 as an alternative to lutetium-177. The aim of the present study was to compare the tolerability of 161Tb- and 177Lu-based DOTA-LM3 and DOTATATE in immunocompetent mice. Dosimetry calculations were performed based on biodistribution data of the radiopeptides in immunocompetent mice. Treatment-related effects on blood cell counts were assessed on Days 10, 28 and 56 after application of [161Tb]Tb-DOTA-LM3 or [161Tb]Tb-DOTATATE at 20 MBq per mouse. These radiopeptides were also applied at 100 MBq per mouse and the effects compared to those observed after application of the 177Lu-labeled counterparts. Bone marrow smears, blood plasma parameters and organ histology were assessed at the end of the study. The absorbed organ dose was commonly higher for the SSTR antagonist than for the SSTR agonist and for terbium-161 over lutetium-177. Application of a therapeutic activity level of 20 MBq [161Tb]Tb-DOTA-LM3 or [161Tb]Tb-DOTATATE was well tolerated without major hematological changes. The injection of 100 MBq of the 161Tb- and 177Lu-based somatostatin analogues affected the blood cell counts, however. The lymphocytes were 40-50% lower in treated mice compared to the untreated controls on Day 10 irrespective of the radionuclide employed. At the same timepoint, thrombocyte and erythrocyte counts were 30-50% and 6-12% lower, respectively, after administration of the SSTR antagonist (p < 0.05) while changes were less pronounced in mice injected with the SSTR agonist. All blood cell counts were in the normal range on Day 56. Histological analyses revealed minimal abnormalities in the kidneys, liver and spleen of treated mice. No correlation was observed between the organ dose and frequency of the occurrence of abnormalities. Hematologic changes were more pronounced in mice treated with the SSTR antagonist than in those treated with the SSTR agonist. Despite the increased absorbed dose delivered by terbium-161 over lutetium-177, [161Tb]Tb-DOTA-LM3 and [161Tb]Tb-DOTATATE should be safe at activity levels that are recommended for their respective 177Lu-based analogues.

Three-dimensional printing in orthopaedic surgery: A review of current and future applications

Three-dimensional (3D) printing is a form of technology in which 3D physical models are created. It has been used in a variety of surgical specialities ranging from cranio-maxillo-facial to orthopaedic surgery and is currently an area of much interest within the medical profession. Within the field of orthopaedic surgery, 3D printing has several clinical applications including surgical education, surgical planning, manufacture of patient-specific prostheses/patient specific instruments and bone tissue engineering. This article reviews the current practices of 3D printing in orthopaedic surgery in both clinical and pre-clinical settings along with discussing its potential future applications.

A gut microbial metabolite cocktail fights against obesity through modulating the gut microbiota and hepatic leptin signaling.

Excessive body weight and obesity elevate the risk of chronic non-communicable diseases. The judicious application of the gut microbiome, encompassing both microorganisms and their derived compounds, holds considerable promise in the treatment of obesity. In this study, we showed that a cocktail of gut microbiota-derived metabolites, comprising indole 3-propionic acid (IPA), sodium butyrate (SB) and valeric acid (VA), alleviated various symptoms of obesity in both male and female mice subjected to a high-fat diet (HFD). The 16S ribosomal RNA (rRNA) sequencing revealed that administering the cocktail via oral gavage retained the gut microbiota composition in obese mice. Fecal microbiota transplantation using cocktail-treated mice as donors mitigated the obesity phenotype of HFD-fed mice. Transcriptomic sequencing analysis showed that the cocktail preserved the gene expression profile of hepatic tissues in obese mice, especially up-regulated the expression level of leptin receptor. Gene delivery via in vivo fluid dynamics further validated that the anti-obesity efficacy of the cocktail was dependent on leptin signaling at least partly. The cocktail also inhibited the expression of appetite stimulators in hypothalamus. Together, the metabolite cocktail combated adiposity by retaining the gut microbiota configuration and activating the hepatic leptin signaling pathway. Our findings provide a sophisticated regulatory network between the gut microbiome and host, and highlight a cocktail of gut microbiota-derived metabolites, including IPA, SB, and VA, might be a prospective intervention for anti-obesity in a preclinical setting. © 2024 Society of Chemical Industry.

Predictors of hip fracture in 15 European countries: a longitudinal study of 48,533 geriatric adults using SHARE dataset.

Hip fracture is a major cause of functional disability, mortality, and health costs. However, the identification and characterization of its causative factors remain poor. We investigated demography, handgrip strength (HGS), depression, and multiple age-associated comorbidities for predicting future hip fracture in individuals aged 50 or above from 15 European countries (n = 48,533). All participants were evaluated from 2013 to 2020 using four successive waves of the Survey of Health, Aging, and Retirement in Europe (SHARE). Altogether, 1130 participants developed hip fractures during the study period. We identified female gender, an advancing age from quinquagenarians onward, and a poor socioeconomic status as critical risk factors for future hip fracture. Having mobility difficulty, a low HGS (< 27kg in men, < 16kg in women) and higher scores on Euro-D depression scales were also significant risk factors for hip fracture. Summated scales of hypertension, diabetes mellitus, cancer, Alzheimer's disease, and stroke did not appear as risk factors. Collectively, we report advancing age, female gender, low HGS, and depression as independent risk factors for hip fracture. Our findings are useful in identifying high-risk populations for hip fractures in pre-clinical settings before rigorous evaluation and treatment in clinics.

Development of Rapid Disk Diffusion Device Using Laser Speckle Formation Technology for Rapid Antimicrobial Susceptibility Testing

The escalation of antimicrobial resistance (AMR) due to the excessive and inappropriate use of antimicrobials has prompted the urgent need for more rapid and effective antimicrobial susceptibility testing (AST) methods. Conventional AST techniques often take 16–24 h, leading to empirical prescription practices and the potential emergence of AMR. The study aimed to develop a rapid disk diffusion (RDD) method utilizing laser speckle formation (LSF) technology to expedite AST results. The study aimed to evaluate the performance of LSF technology in determining antimicrobial susceptibility. In this study, preclinical and clinical settings were established to compare the LSF technology with conventional disk diffusion (DD) methods to measure the inhibition zones. Preclinical experiments with different bacterial strains demonstrated more than 70% categorical agreement (CA) against most antimicrobials. Further, clinical experiments with multiple strains and antibiotics revealed CA ranging from 40 to 79%, while major and minor discrepancies were observed around 30% and 11%, respectively. These observations revealed high concordance between RDD and DD for multiple antimicrobials in multiple species. The results underscore the potential of RDD-based LSF technology for hastening AST procedures. The current study is marked by a unique equipment setup and analysis approach. Collectively, the suggested laser-based RDD showed greater potential than previously developed comparable methods. The proposed method and design have a higher application potential than formerly developed similar technologies. Together, the study contributes to the ongoing development of rapid AST methods.

The Variation in the Traits Ameliorated by Inhibitors of JAK1/2, TGF-β, P-Selectin, and CXCR1/CXCR2 in the Gata1low Model Suggests That Myelofibrosis Should Be Treated by These Drugs in Combination.

Studies conducted on animal models have identified several therapeutic targets for myelofibrosis, the most severe of the myeloproliferative neoplasms. Unfortunately, many of the drugs which were effective in pre-clinical settings had modest efficacy when tested in the clinic. This discrepancy suggests that treatment for this disease requires combination therapies. To rationalize possible combinations, the efficacy in the Gata1low model of drugs currently used for these patients (the JAK1/2 inhibitor Ruxolitinib) was compared with that of drugs targeting other abnormalities, such as p27kip1 (Aplidin), TGF-β (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-β) signaling and TGF-β trap AVID200), P-selectin (RB40.34), and CXCL1 (Reparixin, inhibiting the CXCL1 receptors CXCR1/2). The comparison was carried out by expressing the endpoints, which had either already been published or had been retrospectively obtained for this study, as the fold change of the values in the corresponding vehicles. In this model, only Ruxolitinib was found to decrease spleen size, only Aplidin and SB431542/AVID200 increased platelet counts, and with the exception of AVID200, all the inhibitors reduced fibrosis and microvessel density. The greatest effects were exerted by Reparixin, which also reduced TGF-β content. None of the drugs reduced osteopetrosis. These results suggest that future therapies for myelofibrosis should consider combining JAK1/2 inhibitors with drugs targeting hematopoietic stem cells (p27Kip1) or the pro-inflammatory milieu (TGF-β or CXCL1).

Autotaxin Inhibition Reduces Post-Ischemic Myocardial Inflammation via Epigenetic Gene Modifications.

Myocardial infarction (MI) triggers a complex inflammatory response that is essential for cardiac repair but can also lead to adverse outcomes if left uncontrolled. Recent studies have highlighted the importance of epigenetic modifications in regulating post-MI inflammation. This study investigated the role of the autotaxin (ATX)/lysophosphatidic acid (LPA) signaling axis in modulating myocardial inflammation through epigenetic pathways in a mouse model of MI. C57BL/6J mice underwent left anterior descending coronary artery ligation to induce MI and were treated with the ATX inhibitor, PF-8380, or vehicle. Cardiac tissue from the border zone was collected at 6h, 1, 3, and 7days post-MI for epigenetic gene profiling using RT2 Profiler PCR Arrays. The results revealed distinct gene expression patterns across sham, MI + Vehicle, and MI + PF-8380 groups. PF-8380 treatment significantly altered the expression of genes involved in inflammation, stress response, and epigenetic regulation compared to the vehicle group. Notably, PF-8380 downregulated Hdac5, Prmt5, and Prmt6, which are linked to exacerbated inflammatory responses, as early as 6h post-MI. Furthermore, PF-8380 attenuated the reduction of Smyd1, a gene important in myogenic differentiation, at 7days post-MI. This study demonstrates that the ATX/LPA signaling axis plays a pivotal role in modulating post-MI inflammation via epigenetic pathways. Targeting ATX/LPA signaling may represent a novel therapeutic strategy to control inflammation and improve outcomes after MI. Further research is needed to validate these findings in preclinical and clinical settings and to elucidate the complex interplay between epigenetic mechanisms and ATX/LPA signaling in the context of MI.

Effect of Cangrelor on Infarct Size in ST-Segment-Elevation Myocardial Infarction Treated by Primary Percutaneous Coronary Intervention: A Randomized Controlled Trial (The PITRI Trial).

The administration of intravenous cangrelor at reperfusion achieves faster onset of platelet P2Y12 inhibition than oral ticagrelor and has been shown to reduce myocardial infarction (MI) size in the preclinical setting. We hypothesized that the administration of cangrelor at reperfusion will reduce MI size and prevent microvascular obstruction in patients with ST-segment-elevation MI undergoing primary percutaneous coronary intervention. This was a phase 2, multicenter, randomized, double-blind, placebo-controlled clinical trial conducted between November 2017 to November 2021 in 6 cardiac centers in Singapore. Patients were randomized to receive either cangrelor or placebo initiated before the primary percutaneous coronary intervention procedure on top of oral ticagrelor. The key exclusion criteria included presenting <6 hours of symptom onset; previous MI and stroke or transient ischemic attack; on concomitant oral anticoagulants; and a contraindication for cardiovascular magnetic resonance. The primary efficacy end point was acute MI size by cardiovascular magnetic resonance within the first week expressed as percentage of the left ventricle mass (%LVmass). Microvascular obstruction was identified as areas of dark core of hypoenhancement within areas of late gadolinium enhancement. The primary safety end point was Bleeding Academic Research Consortium-defined major bleeding in the first 48 hours. Continuous variables were compared by Mann-Whitney U test (reported as median [first quartile-third quartile]), and categorical variables were compared by Fisher exact test. A 2-sided P<0.05 was considered statistically significant. Of 209 recruited patients, 164 patients (78%) completed the acute cardiovascular magnetic resonance scan. There were no significant differences in acute MI size (placebo, 14.9% [7.3-22.6] %LVmass versus cangrelor, 16.3 [9.9-24.4] %LVmass; P=0.40) or the incidence (placebo, 48% versus cangrelor, 47%; P=0.99) and extent of microvascular obstruction (placebo, 1.63 [0.60-4.65] %LVmass versus cangrelor, 1.18 [0.53-3.37] %LVmass; P=0.46) between placebo and cangrelor despite a 2-fold decrease in platelet reactivity with cangrelor. There were no Bleeding Academic Research Consortium-defined major bleeding events in either group in the first 48 hours. Cangrelor administered at the time of primary percutaneous coronary intervention did not reduce acute MI size or prevent microvascular obstruction in patients with ST-segment-elevation MI given oral ticagrelor despite a significant reduction of platelet reactivity during the percutaneous coronary intervention procedure. URL: https://www.clinicaltrials.gov; Unique identifier: NCT03102723.

In vivo bioluminescence tomography-guided system for pancreatic cancer radiotherapy research.

Recent development of radiotherapy (RT) has heightened the use of radiation in managing pancreatic cancer. Thus, there is a need to investigate pancreatic cancer in a pre-clinical setting to advance our understanding of the role of RT. Widely-used cone-beam CT (CBCT) imaging cannot provide sufficient soft tissue contrast to guide irradiation. The pancreas is also prone to motion. Large collimation is unavoidably used for irradiation, costing normal tissue toxicity. We innovated a bioluminescence tomography (BLT)-guided system to address these needs. We established an orthotopic pancreatic ductal adenocarcinoma (PDAC) mouse model to access BLT. Mice underwent multi-projection and multi-spectral bioluminescence imaging (BLI), followed by CBCT imaging in an animal irradiator for BLT reconstruction and radiation planning. With optimized absorption coefficients, BLT localized PDAC at 1.25 ± 0.19 mm accuracy. To account for BLT localization uncertainties, we expanded the BLT-reconstructed volume with margin to form planning target volume(PTVBLT) for radiation planning, covering 98.7 ± 2.2% of PDAC. The BLT-guided conformal plan can cover 100% of tumors with limited normal tissue involvement across both inter-animal and inter-fraction cases, superior to the 2D BLI-guided conventional plan. BLT offers unique opportunities to localize PDAC for conformal irradiation, minimize normal tissue involvement, and support reproducibility in RT studies.

Clinical Application of Adenovirus (AdV): A Comprehensive Review.

Adenoviruses are non-enveloped DNA viruses that cause a wide range of symptoms, from mild infections to life-threatening diseases in a broad range of hosts. Due to the unique characteristics of these viruses, they have also become a vehicle for gene-transfer and cancer therapeutic instruments. Adenovirus vectors can be used in gene therapy by modifying wild-type viruses to render them replication-defective. This makes it possible to swap out particular viral genes for segments that carry therapeutic genes and to employ the resultant vector as a means of delivering genes to specified tissues. In this review, we outline the progressive development of adenovirus vectors, exploring their characteristics, genetic modifications, and range of uses in clinical and preclinical settings. A significant emphasis is placed on their crucial role in advancing gene therapy, cancer therapy, immunotherapy, and the latest breakthroughs in vaccine development for various diseases.

Reverse vaccinology approach for identification of epitopes from E1 protein as peptide vaccine against HCV: A proof of concept

The development of effective vaccines against Hepatitis C Virus (HCV) remains a global health priority and challenge. In this study, we employed an integrative approach combining computational epitope prediction with experimental validation to identify immunogenic peptides targeting the E1 glycoprotein of HCV. In the present report, computational data from various epitope prediction algorithms such as IEDB and SYFPEITHI, followed by molecular dynamics (MD) simulations and immuno-informatics analysis is presented. Through computational screening, we identified potential epitope candidates, with QVRNSSGLY (P3) and QLFTFSPRH (P7) emerging as promising candidates. MD simulations revealed stable interactions between these epitopes and MHC molecule, further validated by free energy estimations using MMPBSA method. Immuno-informatics analysis supported these findings, showing high binding potential and immunogenicity scores for the selected peptides. Subsequent synthesis and characterization of epitope peptides confirmed their structural integrity and purity required for conducting immune activation assays. Experimental immunological assays carried out in this study involved epitope peptide induced activation of CD8 + and CD4 + T cells from healthy human subjects and HCV- recovered patients. Data from experimental validation revealed significant cytokine release upon exposure to epitope peptides, particularly TNF-a, IL-6, and GM-CSF, indicative of robust immune responses. Notably, peptides P3 and P7 exhibited the most pronounced cytokine induction profiles, underscoring their potential as vaccine candidates. Further investigations addressing the mechanism of action of these epitope peptides under preclinical and clinical settings may help in developing effective vaccine against HCV.

Timed fetal inflammation and postnatal hypoxia cause cortical white matter injury, interneuron imbalances, and behavioral deficits in a double-hit rat model of encephalopathy of prematurity

Extreme preterm birth-associated adversities are a major risk factor for aberrant brain development, known as encephalopathy of prematurity (EoP), which can lead to long-term neurodevelopmental impairments. Although progress in clinical care for preterm infants has markedly improved perinatal outcomes, there are currently no curative treatment options available to combat EoP. EoP has a multifactorial etiology, including but not limited to pre- or postnatal immune activation and oxygen fluctuations. Elucidating the underlying mechanisms of EoP and determining the efficacy of potential therapies relies on valid, clinically translatable experimental models that reflect the neurodevelopmental and pathophysiological hallmarks of EoP. Here, we expand on our double-hit rat model that can be used to study EoP disease mechanisms and therapeutic options in a preclinical setting. Pregnant Wistar dams were intraperitoneally injected with 10 μg/kg LPS on embryonic day (E)20 and offspring was subjected to hypoxia (140 min, 8% O2) at postnatal day 4. Rats exposed to fetal inflammation and postnatal hypoxia (FIPH) showed neurodevelopmental impairments, such as reduced nest-seeking ability, ultrasonic vocalizations, social engagement, and working memory, and increased anxiety and sensitivity. Impairments in myelination, oligodendrocyte maturation and interneuron development were examined as hallmarks for EoP, in different layers and coordinates of the cortex using histological and molecular techniques. Myelin density and complexity was decreased in the cortex, which partially coincided with a decrease in mature oligodendrocytes. Furthermore, interneuron populations (GAD67+ and PVALB+) were affected. To determine if the timing of inducing fetal inflammation affected the severity of EoP hallmarks in the cortex, multiple timepoints of fetal inflammation were compared. Inflammation at E20 combined with postnatal hypoxia gave the most severe EoP phenotype in the cortex. In conclusion, we present a double-hit rat model which displays various behavioral, anatomical and molecular hallmarks of EoP, including diffuse white matter injury. This double-hit model can be used to investigate pathophysiological mechanisms and potential therapies for EoP.