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Scientific Reports May 2024X-ray Phase Contrast Tomography (XPCT) based on wavefield propagation has been established as a high resolution three-dimensional (3D) imaging modality, suitable to...
X-ray Phase Contrast Tomography (XPCT) based on wavefield propagation has been established as a high resolution three-dimensional (3D) imaging modality, suitable to reconstruct the intricate structure of soft tissues, and the corresponding pathological alterations. However, for biomedical research, more is needed than 3D visualisation and rendering of the cytoarchitecture in a few selected cases. First, the throughput needs to be increased to cover a statistically relevant number of samples. Second, the cytoarchitecture has to be quantified in terms of morphometric parameters, independent of visual impression. Third, dimensionality reduction and classification are required for identification of effects and interpretation of results. To address these challenges, we here design and implement a novel integrated and high throughput XPCT imaging and analysis workflow for 3D histology, pathohistology and drug testing. Our approach uses semi-automated data acquisition, reconstruction and statistical quantification. We demonstrate its capability for the example of lung pathohistology in Covid-19. Using a small animal model, different Covid-19 drug candidates are administered after infection and tested in view of restoration of the physiological cytoarchitecture, specifically the alveolar morphology. To this end, we then use morphometric parameter determination followed by a dimensionality reduction and classification based on optimal transport. This approach allows efficient discrimination between physiological and pathological lung structure, thereby providing quantitative insights into the pathological progression and partial recovery due to drug treatment. Finally, we stress that the XPCT image chain implemented here only used synchrotron radiation for validation, while the data used for analysis was recorded with laboratory CT radiation, more easily accessible for pre-clinical research.
Topics: Animals; COVID-19; Imaging, Three-Dimensional; Lung; SARS-CoV-2; Tomography, X-Ray Computed; Cricetinae; Disease Models, Animal; Drug Evaluation, Preclinical; COVID-19 Drug Treatment
PubMed: 38811688
DOI: 10.1038/s41598-024-61746-4 -
Antibodies (Basel, Switzerland) May 2024The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical...
The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.
PubMed: 38804307
DOI: 10.3390/antib13020039 -
Molecules (Basel, Switzerland) May 2024Disuse muscle atrophy (DMA) is a significant healthcare challenge characterized by progressive loss of muscle mass and function resulting from prolonged inactivity. The...
Disuse muscle atrophy (DMA) is a significant healthcare challenge characterized by progressive loss of muscle mass and function resulting from prolonged inactivity. The development of effective strategies for muscle recovery is essential. In this study, we established a DMA mouse model through hindlimb suspension to evaluate the therapeutic potential of lactate in alleviating the detrimental effects on the gastrocnemius muscle. Using NMR-based metabolomic analysis, we investigated the metabolic changes in DMA-injured gastrocnemius muscles compared to controls and evaluated the beneficial effects of lactate treatment. Our results show that lactate significantly reduced muscle mass loss and improved muscle function by downregulating Murf1 expression, decreasing protein ubiquitination and hydrolysis, and increasing myosin heavy chain levels. Crucially, lactate corrected perturbations in four key metabolic pathways in the DMA gastrocnemius: the biosynthesis of phenylalanine, tyrosine, and tryptophan; phenylalanine metabolism; histidine metabolism; and arginine and proline metabolism. In addition to phenylalanine-related pathways, lactate also plays a role in regulating branched-chain amino acid metabolism and energy metabolism. Notably, lactate treatment normalized the levels of eight essential metabolites in DMA mice, underscoring its potential as a therapeutic agent against the consequences of prolonged inactivity and muscle wasting. This study not only advances our understanding of the therapeutic benefits of lactate but also provides a foundation for novel treatment approaches aimed at metabolic restoration and muscle recovery in conditions of muscle wasting.
Topics: Animals; Mice; Metabolomics; Lactic Acid; Muscle, Skeletal; Muscular Atrophy; Disease Models, Animal; Magnetic Resonance Spectroscopy; Male; Muscle Proteins; Muscular Disorders, Atrophic; Ubiquitin-Protein Ligases; Metabolome; Hindlimb Suspension; Tripartite Motif Proteins; Mice, Inbred C57BL; Myosin Heavy Chains
PubMed: 38792078
DOI: 10.3390/molecules29102216 -
Phytomedicine : International Journal... Jul 2024Cerebral ischemia-reperfusion injury (CIRI) refers to brain tissue injury caused by the temporary interruption of cerebral blood flow ischemia followed by the...
BACKGROUND
Cerebral ischemia-reperfusion injury (CIRI) refers to brain tissue injury caused by the temporary interruption of cerebral blood flow ischemia followed by the restoration of reperfusion, which is the main cause of post-stroke brain injury. A traditional Chinese herbal preparation called Tongqiao Huoxue Decoction (TQHX) has shown promise in reducing CIRI in rats. However, the mechanism of this herbal preparation for CIRI remains unclear.
PURPOSE
This study aimed to evaluate the therapeutic effect of TQHX extract on rats with CIRI and to further explore the underlying mechanisms.
METHODS
The active ingredients of TQHX extract were quantified by the high-performance liquid chromatography (HPLC) condition. We conducted thorough investigations to assess the effects of TQHX on CIRI and ferroptosis using oxygen-glucose deprivation/reperfusion (OGD/R)-treated PC12 cells as an in vitro model and transient middle cerebral artery occlusion (tMCAO) animals as an in vivo model. The neurological score assessment was performed to evaluate the neuroprotective effects of TQHX extract on tMCAO rats. Using histologic methods to study the extent of cerebral infarction, blood-brain barrier, and rat brain tissue. We examined the impact of TQHX on ferroptosis-related markers of Fe, superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) in the brain tissue. In addition, the expression of key proteins and markers of ferroptosis, as well as key factors associated with Acyl-CoA synthetase long-chain family member 4 (ACSL4) were detected by Western blot and quantitative real-time PCR (RT-qPCR).
RESULTS
TQHX extract could decrease the Longa score and extent of cerebral infarction of tMCAO rats, which exerted the function of neuroprotection. Additionally, TQHX treatment efficiently decreased levels of MDA and ROS while increasing the expression of SOD and ferroptosis-related proteins including ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) at the transcription and translation level. Meanwhile, TQHX provided strong protection against oxidative stress and ferritin accumulation by increasing the ubiquitination and degradation of ACSL4. The injection of OE-ACSL4 reversed the effects of TQHX on neuroprotection and ferroptosis inhibition in PC12 cells. The injection of shACSL4 reversely validate the crucial role of ACSL4 in CIRI rat treatment.
CONCLUSION
This work shows that TQHX promotes the ubiquitination-mediated degradation of ACSL4, which improves oxidative stress and inhibits the beginning of ferroptosis in cells. TQHX provides a possible path for additional research in CIRI therapies, advancing translational investigations.
Topics: Animals; Male; Rats; Brain Ischemia; Coenzyme A Ligases; Disease Models, Animal; Drugs, Chinese Herbal; Ferroptosis; Infarction, Middle Cerebral Artery; Neuroprotective Agents; Oxidative Stress; PC12 Cells; Rats, Sprague-Dawley; Reperfusion Injury; Ubiquitination
PubMed: 38788392
DOI: 10.1016/j.phymed.2024.155701 -
Molecular Medicine Reports Jul 2024Promotion of myoblast differentiation by activating mitochondrial biogenesis and protein synthesis signaling pathways provides a potential alternative strategy to...
Promotion of myoblast differentiation by activating mitochondrial biogenesis and protein synthesis signaling pathways provides a potential alternative strategy to balance energy and overcome muscle loss and muscle disorders. (Lour.) Baill. extract (SCE) has been used extensively as a traditional herbal medicine and has several physiological activities, including anti‑asthmatic, anti‑oxidant, anti‑inflammatory, anti‑atopic, anticancer and hepatoprotective properties. However, the effects and mechanisms of action of SCE on muscle differentiation have not yet been clarified. In the present study, it was investigated whether SCE affects skeletal muscle cell differentiation through the regulation of mitochondrial biogenesis and protein synthesis in murine C2C12 myoblasts. The XTT colorimetric assay was used to determine cell viability, and myosin heavy chain (MyHC) levels were determined using immunocytochemistry. SCE was applied to C2C12 myotube at different concentrations (1, 5, or 10 ng/ml) and times (1,3, or 5 days). Reverse transcription‑quantitative PCR and western blotting were used to analyze the mRNA and protein expression change of factors related to differentiation, mitochondrial biogenesis and protein synthesis. Treatment of C2C12 cells with SCE at 1,5, and 10 ng/ml did not affect cell viability. SCE promoted C2C12 myotube formation and significantly increased MyHC expression in a concentration‑ and time‑dependent manner. SCE significantly increased the mRNA and protein expression of muscle differentiation‑specific markers, such as MyHC, myogenic differentiation 1, myogenin, Myogenic Factor 5, and β‑catenin, mitochondrial biosynthesis‑related factors, such as peroxisome proliferator‑activated receptor‑gamma coactivator‑1α, nuclear respirator factor‑1, AMP‑activated protein kinase phosphorylation, and histone deacetylase 5 and AKT/mTOR signaling factors related to protein synthesis. SCE may prevent skeletal muscle dysfunction by enhancing myoblast differentiation through the promotion of mitochondrial biogenesis and protein synthesis.
Topics: Animals; Mice; Cell Differentiation; Signal Transduction; TOR Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Plant Extracts; Organelle Biogenesis; Cell Line; Saururaceae; Cell Survival; Myoblasts; Mitochondria; Muscle Development; Muscle Fibers, Skeletal; Myosin Heavy Chains; Muscle, Skeletal
PubMed: 38785149
DOI: 10.3892/mmr.2024.13250 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein....
In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.
Topics: Animals; Female; Mice; Antibodies, Monoclonal; Antibodies, Viral; Escherichia coli; Hybridomas; Mice, Inbred BALB C; Nucleocapsid Proteins; Orthobunyavirus; Recombinant Proteins
PubMed: 38783815
DOI: 10.13345/j.cjb.230731 -
Lower Urinary Tract Symptoms May 2024This study evaluates the impact of equol, a metabolite of soy isoflavone, on bladder dysfunction in rats with bladder outlet obstruction (BOO). In addition, we...
OBJECTIVES
This study evaluates the impact of equol, a metabolite of soy isoflavone, on bladder dysfunction in rats with bladder outlet obstruction (BOO). In addition, we investigate its potential as a neuroprotective agent for the obstructed bladder and discuss its applicability in managing overactive bladder (OAB).
METHODS
Eighteen male Sprague-Dawley rats were divided into three groups (six rats per group) during the rearing period. The Sham and C-BOO groups received an equol-free diet, while the E-BOO group received equol supplementation (0.25 g/kg). At 8 weeks old, rats underwent BOO surgery, followed by continuous cystometry after 4 weeks of rearing. The urinary oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde) were measured, and the bladder histology was analyzed using hematoxylin-eosin, Masson's trichrome, and immunohistochemical staining (neurofilament heavy chain for myelinated nerves, peripherin for unmyelinated nerves, and malondialdehyde).
RESULTS
Equol reduced BOO-induced smooth muscle layer fibrosis, significantly prolonged the micturition interval (C-BOO: 193 s, E-BOO: 438 s) and increased the micturition volume (C-BOO: 0.54 mL, E-BOO: 1.02 mL) compared to the C-BOO group. Equol inhibited the increase in urinary and bladder tissue malondialdehyde levels. While the C-BOO group exhibited reduced peripherin alone positive nerve fibers within the smooth muscle layer, equol effectively attenuated this decline.
CONCLUSIONS
Equol reduces lipid peroxidation and smooth muscle layer fibrosis in the bladder and exhibited neuroprotective effects on bladder nerves (peripheral nerves) and prevented the development of bladder dysfunction associated with BOO in rats. Consumption of equol is promising for the prevention of OAB associated with BOO.
Topics: Animals; Rats, Sprague-Dawley; Male; Equol; Urinary Bladder Neck Obstruction; Rats; Urinary Bladder; Oxidative Stress; Disease Models, Animal; Urinary Bladder, Overactive; Malondialdehyde; Neuroprotective Agents; Urination; Fibrosis
PubMed: 38777796
DOI: 10.1111/luts.12518 -
Journal of Animal Science Jan 2024Both n-6 and n-3 fatty acids (FA) have numerous significant physiological roles for mammals. The interplay between these families of FA is of interest in companion... (Review)
Review
Both n-6 and n-3 fatty acids (FA) have numerous significant physiological roles for mammals. The interplay between these families of FA is of interest in companion animal nutrition due to the influence of the n-6:n-3 FA ratio on the modulation of the inflammatory response in disease management and treatment. As both human and animal diets have shifted to greater consumption of vegetable oils rich in n-6 FA, the supplementation of n-3 FA to canine, feline, and equine diets has been advocated for. Although fish oils are commonly added to supply the long-chain n-3 FA eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), a heavy reliance on this ingredient by the human, pet food, and equine supplement industries is not environmentally sustainable. Instead, sustainable sourcing of plant-based oils rich in n-3 α-linolenic acid (ALA), such as flaxseed and camelina oils, emerges as a viable option to support an optimal n-6:n-3 FA ratio. Moreover, ALA may offer health benefits that extend beyond its role as a precursor for endogenous EPA and DHA production. The following review underlines the metabolism and recommendations of n-6 and n-3 FA for dogs, cats, and horses and the ratio between them in promoting optimal health and inflammation management. Additionally, insights into both marine and plant-based n-3 FA sources will be discussed, along with the commercial practicality of using plant oils rich in ALA for the provision of n-3 FA to companion animals.
Topics: Animals; Dogs; Horses; Cats; Fatty Acids, Omega-3; alpha-Linolenic Acid; Fatty Acids, Omega-6; Animal Feed; Diet; Animal Nutritional Physiological Phenomena
PubMed: 38776363
DOI: 10.1093/jas/skae143 -
Annals of Clinical and Translational... May 2024Neurofilament heavy-chain gene (NEFH) variants are associated with multiple neurodegenerative diseases, however, their relationship with ALS has not been robustly...
OBJECTIVE
Neurofilament heavy-chain gene (NEFH) variants are associated with multiple neurodegenerative diseases, however, their relationship with ALS has not been robustly explored. Still, NEFH is commonly included in genetic screening panels worldwide. We therefore aimed to determine if NEFH variants modify ALS risk.
METHODS
Genetic data of 11,130 people with ALS and 7,416 controls from the literature and Project MinE were analysed. We performed meta-analyses of published case-control studies reporting NEFH variants, and variant analysis of NEFH in Project MinE whole-genome sequencing data.
RESULTS
Fixed-effects meta-analysis found that rare (MAF <1%) missense variants in the tail domain of NEFH increase ALS risk (OR 4.55, 95% CI 2.13-9.71, p < 0.0001). In Project MinE, ultrarare NEFH variants increased ALS risk (OR 1.37 95% CI 1.14-1.63, p = 0.0007), with rod domain variants (mostly intronic) appearing to drive the association (OR 1.45 95% CI 1.18-1.77, p = 0.0007, p = 0.003). While in the tail domain, ultrarare (MAF <0.1%) pathogenic missense variants were also associated with higher risk of ALS (OR 1.94, 95% CI 0.86-4.37, p = 0.039), supporting the meta-analysis results. Finally, several tail in-frame deletions were also found to affect disease risk, however, both protective and pathogenic deletions were found in this domain, highlighting an intricated architecture that requires further investigation.
INTERPRETATION
We showed that NEFH tail missense and in-frame deletion variants, and intronic rod variants are risk factors for ALS. However, they are not variants of large effect, and their functional impact needs to be clarified in further studies. Therefore, their inclusion in routine genetic screening panels should be reconsidered.
PubMed: 38775181
DOI: 10.1002/acn3.52083 -
PloS One 2024Determining SNP-SNP interaction of the disease has become important for further investigation of pathogenesis and experimental research. Although many studies have been...
Determining SNP-SNP interaction of the disease has become important for further investigation of pathogenesis and experimental research. Although many studies have been published on the effect of MMPs gene polymorphisms on chronic obstructive pulmonary disease (COPD), there is a lack of information on SNP-SNP and SNP-environment interactions. This study aimed to investigate the interaction between the polymorphisms of MMP1, MMP2, MMP9 and MMP12 genes and its combined effect with smoking on the risk of developing COPD. Totally 181 COPD patients and 292 healthy individuals were involved. Blood samples from the participants were tested for genotyping and data were collected through questionnaires. Genotyping was performed with nested allele-specific polymerase chain reaction (AS-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). SNP-SNP and SNP-environment interactions were investigated using multifactor dimensionality reduction and logistic regression analysis. The result showed that participants with high nicotine dependence and heavy smokers had a higher risk of COPD than non-smokers. Also, G/G genotype (cOR = 5.83; 95% CI, 1.19-28.4, p = 0.029) of MMP2 rs243864 and T/T genotype (cOR = 1.79; 95% CI, 1.16-2.76, p = 0.008) of MMP12 rs652438 independently contributes to the susceptibility of COPD. For SNP-SNP interaction, the positive interaction between rs243864 G/G genotype of MMP2 and rs652438 T/T genotype of MMP12 was found, and the combination of risk genotypes has a high risk of COPD (OR = 12.92; 95% CI, 1.46-114.4, p = 0.021). Moreover, the combination of T/T genotype of MMP12 rs652438 and smoking-related factors increases the risk of COPD approximately 4.5 to 6-fold. The results suggests that there is a combination of MMP2, MMP12, and smoking-related factors may increase the risk of developing COPD.
Topics: Humans; Pulmonary Disease, Chronic Obstructive; Polymorphism, Single Nucleotide; Matrix Metalloproteinase 12; Male; Female; Matrix Metalloproteinase 2; Middle Aged; Genetic Predisposition to Disease; Aged; Case-Control Studies; Smoking; Genotype; Risk Factors
PubMed: 38771844
DOI: 10.1371/journal.pone.0301807