Inhibition of SARS-CoV-2 Nsp9 ssDNA-Binding Activity and Cytotoxic Effects on H838, H1975, and A549 Human Non-Small Cell Lung Cancer Cells: Exploring the Potential of Nepenthes miranda Leaf Extract for Pulmonary Disease Treatment.

Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores the therapeutic potential of Nepenthes miranda leaf extract against nonstructural protein 9 (Nsp9) of SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) cell lines. Nsp9, essential for SARS-CoV-2 RNA replication, was expressed and purified, and its interaction with ssDNA was assessed. Initial tests with myricetin and oridonin, known for targeting ssDNA-binding proteins and Nsp9, respectively, did not inhibit the ssDNA-binding activity of Nsp9. Subsequent screenings of various N. miranda extracts identified those using acetone, methanol, and ethanol as particularly effective in disrupting Nsp9's ssDNA-binding activity, as evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol and lupenone, major components in the leaf extract of N. miranda, as potential inhibitors. The cytotoxic properties of N. miranda leaf extract were examined across NSCLC lines H1975, A549, and H838, focusing on cell survival, apoptosis, and migration. Results showed a dose-dependent cytotoxic effect in the following order: H1975 > A549 > H838 cells, indicating specificity. Enhanced anticancer effects were observed when the extract was combined with afatinib, suggesting synergistic interactions. Flow cytometry indicated that N. miranda leaf extract could induce G2 cell cycle arrest in H1975 cells, potentially inhibiting cancer cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 19 most abundant compounds in the leaf extract of N. miranda. These outcomes underscore the dual utility of N. miranda leaf extract in potentially managing SARS-CoV-2 infection through Nsp9 inhibition and offering anticancer benefits against lung carcinoma. These results significantly broaden the potential medical applications of N. miranda leaf extract, suggesting its use not only in traditional remedies but also as a prospective treatment for pulmonary diseases. Overall, our findings position the leaf extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and antiviral therapies, warranting further investigation into its molecular mechanisms and potential clinical applications.

Oral Frenum Composition and Clinical Implications in Bone Regeneration: A Review.

Successful bone augmentation relies on primary wound closure. The labial frenum is a soft tissue that connects the lip to the alveolar mucosa or gingiva. However, the frenum may exert biomechanical forces to the wound edge, causing wound instability. The aim of this study is to review the frenum composition and classifications and to understand the significance of the frenum in wound stability upon bone regeneration. Together with a manual search, an electronic search was conducted through three online databases on studies published until September 2022. A total of 300 articles were identified, and 9 studies were included in this review. Two of the included studies discovered that 35% to 37.5% of the labial frenum had muscle fibers. Other studies showed that the labial frenum was mainly composed of connective tissue with elastic fibers. There are two widely used classifications for the frenum based on its morphology and attachment position. No studies specifically evaluated the impact of the frenum on bone regeneration, but the frenum location intercorrelated with the amount of keratinized tissue, which could influence wound stability. A modified frenum classification for the edentulous ridge and a decision diagram to manage the frenum is proposed for research and evidence-based practice.

Isolation and Characterization of Insecticidal Cyclotides from Viola communis.

Cyclotides are cysteine-rich plant-derived peptides composed of 28-37 amino acids with a head-to-tail cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Their beneficial biophysical properties make them promising molecules for pharmaceutical and agricultural applications. The Violaceae plant family is the major cyclotide-producing family, and to date, every examined plant from this family has been found to contain cyclotides. The presence of cyclotides in Viola communis was inferred by mass spectroscopy previously, but their sequences and properties had yet to be explored. In this study, the occurrence of cyclotides in this plant was investigated using proteomics and transcriptomics. Twenty cyclotides were identified at the peptide level, including two new members from the bracelet (Vcom1) and Möbius (Vcom2) subfamilies. Structural analysis of these newly identified peptides demonstrated a similar fold compared with cyclotides from the same respective subfamilies. Biological assays of Vcom1 and Vcom2 revealed them to be cytotoxic to Sf9 insect cell lines, with Vcom1 demonstrating higher potency than Vcom2. The results suggest that they could be further explored as insecticidal agents and confirm earlier general findings that bracelet cyclotides have more potent insecticidal activity than their Möbius relatives. Seven new cyclotide-like sequences were observed in the transcriptome of V. communis, highlighting the Violaceae as a rich source for new cyclotides with potential insecticidal activity. An analysis of sequences flanking the cyclotide domain in the various precursors from V. communis and other Violaceae plants revealed new insights into cyclotide processing and suggested the possibility of two alternative classes of N-terminal processing enzymes for cyclotide biosynthesis.

Repurposing a plant peptide cyclase for targeted lysine acylation.

Transpeptidases are powerful tools for protein engineering but are largely restricted to acting at protein backbone termini. Alternative enzymatic approaches for internal protein labelling require bulky recognition motifs or non-proteinogenic reaction partners, potentially restricting which proteins can be modified or the types of modification that can be installed. Here we report a strategy for labelling lysine side chain ε-amines by repurposing an engineered asparaginyl ligase, which naturally catalyses peptide head-to-tail cyclization, for versatile isopeptide ligations that are compatible with peptidic substrates. We find that internal lysines with an adjacent leucine residue mimic the conventional N-terminal glycine-leucine substrate. This dipeptide motif enables efficient intra- or intermolecular ligation through internal lysine side chains, minimally leaving an asparagine C-terminally linked to the lysine side chain via an isopeptide bond. The versatility of this approach is demonstrated by the chemoenzymatic synthesis of peptides with non-native C terminus-to-side chain topology and the conjugation of chemically modified peptides to recombinant proteins.

Identification of Molecular Profile of Ear Fibroblasts Derived from Spindle-Transferred Holstein Cattle with Ooplasts from Taiwan Yellow Cattle under Heat Stress.

Global warming has a significant impact on the dairy farming industry, as heat stress causes reproductive endocrine imbalances and leads to substantial economic losses, particularly in tropical-subtropical regions. The Holstein breed, which is widely used for dairy production, is highly susceptible to heat stress, resulting in a dramatic reduction in milk production during hot seasons. However, previous studies have shown that cells of cows produced from reconstructed embryos containing cytoplasm (o) from Taiwan yellow cattle (Y) have improved thermotolerance despite their nuclei (n) being derived from heat-sensitive Holstein cattle (H). Using spindle transfer (ST) technology, we successfully produced ST-Yo-Hn cattle and proved that the thermotolerance of their ear fibroblasts is similar to that of Y and significantly better than that of H (p < 0.05). Despite these findings, the genes and molecules responsible for the different sensitivities of cells derived from ST-Yo-Hn and H cattle have not been extensively investigated. In the present study, ear fibroblasts from ST-Yo-Hn and H cattle were isolated, and differentially expressed protein and gene profiles were compared with or without heat stress (hs) (42 °C for 12 h). The results revealed that the relative protein expression levels of pro-apoptotic factors, including Caspase-3, -8, and -9, in the ear fibroblasts from the ST-Yo-Hn-hs group were significantly lower (p < 0.05) than those from the H-hs group. Conversely, the relative expression levels of anti-apoptotic factors, including GNA14 protein and the CRELD2 and PRKCQ genes, were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Analysis of oxidative phosphorylation-related factors revealed that the relative expression levels of the GPX1 gene and Complex-I, Complex-IV, CAT, and PGLS proteins were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Taken together, these findings suggest that ear fibroblasts from ST-Yo-Hn cattle have superior thermotolerance compared to those from H cattle due to their lower expression of pro-apoptotic factors and higher expression of oxidative phosphorylation and antioxidant factors. Moreover, this improved thermotolerance is attributed, at least partially, to the cytoplasm derived from more heat-tolerant Y cattle. Hence, using ST technology to produce more heat-tolerant H cattle containing Y cytoplasm could be a feasible approach to alleviate the negative impacts of heat stress on dairy cattle in tropical-subtropical regions.

Potato protein hydrolysate inhibits RANKL-induced osteoclast development by inhibiting osteoclastogenic genes via the NF-κB/MAPKs signaling pathways.

In recent times, there has been growing attention towards exploring the nutritional and functional aspects of potato protein, along with its diverse applications. In the present study, we examined the anti-osteoclast properties of potato protein hydrolysate (PP902) in vitro. Murine macrophages (RAW264.7) were differentiated into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL), and PP902 was examined for its inhibitory effect. Initially, treatment with PP902 was found to significantly prevent RANKL-induced morphological changes in macrophage cells, as determined by tartrate-resistant acid phosphatase (TRAP) staining analysis. This notion was further supported by F-actin analysis using a confocal microscope. Furthermore, PP902 treatment effectively and dose-dependently down-regulated the expression of RANKL-induced osteoclastogenic marker genes, including TRAP, CTR, RANK, NFATc1, OC-STAMP, and c-Fos. These inhibitory effects were associated with suppressing NF-κB transcriptional activation and subsequent reduced nuclear translocation. The decrease in NF-κB activity resulted from reduced activation of its upstream kinases, including I-κBα and IKKα. Moreover, PP902 significantly inhibited RANKL-induced p38MAPK and ERK1/2 activities. Nevertheless, PP902 treatment prevents RANKL-induced intracellular reactive oxygen species generation via increased HO-1 activity. The combined antioxidant and anti-inflammatory effects of PP902 resulted in significant suppression of osteoclastogenesis, suggesting its potential as an adjuvant therapy for osteoclast-related diseases.

Structure and Activity of Reconstructed Pseudo-Ancestral Cyclotides.

Cyclotides are cyclic peptides that are promising scaffolds for the design of drug candidates and chemical tools. However, despite there being hundreds of reported cyclotides, drug design studies have commonly focussed on a select few prototypic examples. Here, we explored whether ancestral sequence reconstruction could be used to generate new cyclotides for further optimization. We show that the reconstructed 'pseudo-ancestral' sequences, named Ancy-m (for the ancestral cyclotide of the Möbius sub-family) and Ancy-b (for the bracelet sub-family), have well-defined structures like their extant members, comprising the core structural feature of a cyclic cystine knot. This motif underpins efforts to re-engineer cyclotides for agrochemical and therapeutic applications. We further show that the reconstructed sequences are resistant to temperatures approaching boiling, bind to phosphatidyl-ethanolamine lipid bilayers at micromolar affinity, and inhibit the growth of insect cells at inhibitory concentrations in the micromolar range. Interestingly, the Ancy-b cyclotide had a higher oxidative folding yield than its comparator cyclotide cyO2, which belongs to the bracelet cyclotide subfamily known to be notoriously difficult to fold. Overall, this study provides new cyclotide sequences not yet found naturally that could be valuable starting points for the understanding of cyclotide evolution and for further optimization as drug leads.

Cytotoxicity and Multi-Enzyme Inhibition of Nepenthes miranda Stem Extract on H838 Human Non-Small Cell Lung Cancer Cells and RPA32, Elastase, Tyrosinase, and Hyaluronidase Proteins.

The carnivorous pitcher plants of the genus Nepenthes have long been known for their ethnobotanical applications. In this study, we prepared various extracts from the pitcher, stem, and leaf of Nepenthes miranda using 100% ethanol and assessed their inhibitory effects on key enzymes related to skin aging, including elastase, tyrosinase, and hyaluronidase. The cytotoxicity of the stem extract of N. miranda on H838 human lung carcinoma cells were also characterized by effects on cell survival, migration, proliferation, apoptosis induction, and DNA damage. The cytotoxic efficacy of the extract was enhanced when combined with the chemotherapeutic agent 5-fluorouracil (5-FU), indicating a synergistic effect. Flow cytometry analysis suggested that the stem extract might suppress H838 cell proliferation by inducing G2 cell cycle arrest, thereby inhibiting carcinoma cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 15 most abundant compounds in the stem extract of N. miranda. Notably, the extract showed a potent inhibition of the human RPA32 protein (huRPA32), critical for DNA replication, suggesting a novel mechanism for its anticancer action. Molecular docking studies further substantiated the interaction between the extract and huRPA32, highlighting bioactive compounds, especially the two most abundant constituents, stigmast-5-en-3-ol and plumbagin, as potential inhibitors of huRPA32's DNA-binding activity, offering promising avenues for cancer therapy. Overall, our findings position the stem extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and anti-skin-aging treatments, warranting further investigation into its molecular mechanisms and potential clinical applications.

Season of death, pathogen persistence and wildlife behaviour alter number of anthrax secondary infections from environmental reservoirs.

An important part of infectious disease management is predicting factors that influence disease outbreaks, such as R, the number of secondary infections arising from an infected individual. Estimating R is particularly challenging for environmentally transmitted pathogens given time lags between cases and subsequent infections. Here, we calculated R for Bacillus anthracis infections arising from anthrax carcass sites in Etosha National Park, Namibia. Combining host behavioural data, pathogen concentrations and simulation models, we show that R is spatially and temporally variable, driven by spore concentrations at death, host visitation rates and early preference for foraging at infectious sites. While spores were detected up to a decade after death, most secondary infections occurred within 2 years. Transmission simulations under scenarios combining site infectiousness and host exposure risk under different environmental conditions led to dramatically different outbreak dynamics, from pathogen extinction (R < 1) to explosive outbreaks (R > 10). These transmission heterogeneities may explain variation in anthrax outbreak dynamics observed globally, and more generally, the critical importance of environmental variation underlying host-pathogen interactions. Notably, our approach allowed us to estimate the lethal dose of a highly virulent pathogen non-invasively from observational studies and epidemiological data, useful when experiments on wildlife are undesirable or impractical.

Delivery to, and Reactivation of, the p53 Pathway in Cancer Cells Using a Grafted Cyclotide Conjugated with a Cell-Penetrating Peptide.

Peptides are promising drug modalities that can modulate protein-protein interactions, but their application is hampered by their limited ability to reach intracellular targets. Here, we improved the cytosolic delivery of a peptide blocking p53:MDM2/X interactions using a cyclotide as a stabilizing scaffold. We applied several design strategies to improve intracellular delivery and found that the conjugation of the lead cyclotide to the cyclic cell-penetrating peptide cR10 was the most effective. Conjugation allowed cell internalization at micromolar concentration and led to elevated intracellular p53 levels in A549, MCF7, and MCF10A cells, as well as inducing apoptosis in A549 cells without causing membrane disruption. The lead peptide had >35-fold improvement in inhibitory activity and increased cellular uptake compared to a previously reported cyclotide p53 activator. In summary, we demonstrated the delivery of a large polar cyclic peptide in the cytosol and confirmed its ability to modulate intracellular protein-protein interactions involved in cancer.

Molecular dynamics simulations support a preference of cyclotide kalata B1 for phosphatidylethanolamine phospholipids.

Kalata B1 (kB1), a naturally occurring cyclotide has been shown experimentally to bind lipid membranes that contain phosphatidylethanolamine (PE) phospholipids. Here, molecular dynamics simulations were used to explore its interaction with two phospholipids, palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), and a heterogeneous membrane comprising POPC/POPE (90:10), to understand the basis for the selectivity of kB1 towards PE phospholipids. The simulations showed that in the presence of only 10 % POPE lipid, kB1 forms a stable binding complex with membrane bilayers. An ionic interaction between the E7 carboxylate group of kB1 and the ammonium group of PE headgroups consistently initiates binding of kB1 to the membrane. Additionally, stable noncovalent interactions such as hydrogen bonding (E7, T8, V10, G11, T13 and N15), cation-π (W23), and CH-π (W23) interactions between specific residues of kB1 and the lipid membrane play an important role in stabilizing the binding. These findings are consistent with a structure-activity relationship study on kB1 where lysine mutagenesis on the bioactive and hydrophobic faces of the peptide abolished membrane-dependent bioactivities. In summary, our simulations suggest the importance of residue E7 (in the bioactive face) in enabling kB1 to recognize and bind selectively to PE-containing phospholipids bilayers through ionic and hydrogen bonding interactions, and of W23 (in the hydrophobic face) for the association and insertion of kB1 into the lipid bilayer through cation-π and CH-π interactions. Overall, this work enhances our understanding of the molecular basis of the membrane binding and bioactivity of this prototypic cyclotide.

Scanning mutagenesis identifies residues that improve the long-term stability and insecticidal activity of cyclotide kalata B1.

Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of ∼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.

Critical roles of tubular mitochondrial ATP synthase dysfunction in maleic acid-induced acute kidney injury.

Maleic acid (MA) induces renal tubular cell dysfunction directed to acute kidney injury (AKI). AKI is an increasing global health burden due to its association with mortality and morbidity. However, targeted therapy for AKI is lacking. Previously, we determined mitochondrial-associated proteins are MA-induced AKI affinity proteins. We hypothesized that mitochondrial dysfunction in tubular epithelial cells plays a critical role in AKI. In vivo and in vitro systems have been used to test this hypothesis. For the in vivo model, C57BL/6 mice were intraperitoneally injected with 400 mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2 mM or 5 mM MA for 24 h. AKI can be induced by administration of MA. In the mice injected with MA, the levels of blood urea nitrogen (BUN) and creatinine in the sera were significantly increased (p < 0.005). From the pathological analysis, MA-induced AKI aggravated renal tubular injuries, increased kidney injury molecule-1 (KIM-1) expression and caused renal tubular cell apoptosis. At the cellular level, mitochondrial dysfunction was found with increasing mitochondrial reactive oxygen species (ROS) (p < 0.001), uncoupled mitochondrial respiration with decreasing electron transfer system activity (p < 0.001), and decreasing ATP production (p < 0.05). Under transmission electron microscope (TEM) examination, the cristae formation of mitochondria was defective in MA-induced AKI. To unveil the potential target in mitochondria, gene expression analysis revealed a significantly lower level of ATPase6 (p < 0.001). Renal mitochondrial protein levels of ATP subunits 5A1 and 5C1 (p < 0.05) were significantly decreased, as confirmed by protein analysis. Our study demonstrated that dysfunction of mitochondria resulting from altered expression of ATP synthase in renal tubular cells is associated with MA-induced AKI. This finding provides a potential novel target to develop new strategies for better prevention and treatment of MA-induced AKI.

The complexed crystal structure of dihydropyrimidinase reveals a potential interactive link with the neurotransmitter γ-aminobutyric acid (GABA).

Dihydropyrimidinase (DHPase) plays a crucial role in pyrimidine degradation, showcasing a broad substrate specificity that extends beyond pyrimidine catabolism, hinting at additional roles for this ancient enzyme. In this study, we solved the crystal structure of Pseudomonas aeruginosa DHPase (PaDHPase) complexed with the neurotransmitter γ-aminobutyric acid (GABA) at a resolution of 1.97 Å (PDB ID 8WQ9). Our structural analysis revealed two GABA binding sites in each monomer of PaDHPase. Interactions between PaDHPase and GABA molecules, involving residues within a contact distance of <4 Å, were examined. In silico analyses via PISA and PLIP software revealed hydrogen bonds formed between the side chain of Cys318 and GABA 1, as well as the main chains of Ser333, Ile335, and Asn337 with GABA 2. Comparative structural analysis between GABA-bound and unbound states unveiled significant conformational changes at the active site, particularly within dynamic loop I, supporting the conclusion that PaDHPase binds GABA through the loop-out mechanism. Building upon this molecular evidence, we discuss and propose a working model. The study expands the GABA interactome by identifying DHPase as a novel GABA-interacting protein and provides structural insight into the interaction between a dimetal center in the protein's active site and GABA. Further investigations are warranted to explore potential interactions of GABA with other DHPase-like proteins and to understand whether DHPase may have additional regulatory and physiological roles in the cell, extending beyond pyrimidine catabolism.

The power of knowledge: How mental health literacy can overcome barriers to seeking help.

Mental health literacy (MHL) predicts help-seeking attitudes. However, the relationship between components of MHL and help-seeking attitudes has not been thoroughly examined. This study aims to examine whether mental illness stigma, help-seeking efficacy, and maintenance of positive mental health mediated the relationship between recognition of mental disorders and help-seeking attitudes, using a meta-analytic structural equation modeling (MASEM) approach. A comprehensive literature search was conducted to gather relevant studies (111 articles with 118 independent samples), and their data (k = 185) were analyzed using MASEM. Reducing mental illness stigma or increasing help-seeking efficacy may be effective strategies for promoting help-seeking behaviors among individuals who recognize mental disorders, while the maintenance of positive mental health did not significantly mediate the relationship between recognition of mental disorders and help-seeking attitudes. These findings suggest that reducing stigma or increasing help-seeking efficacy may be an effective strategy for promoting help-seeking behaviors among individuals who can identify mental disorders. The use of MASEM in this study highlights the importance of integrating multiple studies to understand the complex relationship between MHL components and help-seeking attitudes. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Computational Design of α-Conotoxins to Target Specific Nicotinic Acetylcholine Receptor Subtypes.

Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α-conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high-resolution experimental structures, which are not widely available for α-conotoxin complexes. Herein, five mutational energy prediction methods were benchmarked using crystallographic and mutational data on two acetylcholine binding protein/α-conotoxin systems. Molecular models were developed for six nAChR subtypes in complex with five α-conotoxins that were studied through 150 substitutions. The best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85 % accuracy). Novel α-conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype-specific nAChR ligands and potentially accelerated drug development.

Antiviral therapy reduces hepatocellular carcinoma through suppressing hepatitis B virus replication may improve ER stress, mitochondrial and metabolic dysfunctions and decrease p62 in hybridized mice with single HBV transgene and miR-122.

Hepatitis B virus (HBV) hijacks autophagy for its replication. Nucleos(t)ide analogs (NUCs) treatment suppressed HBV replication and reduced hepatocellular carcinoma (HCC) incidence. However, the use of NUCs in chronic hepatitis B (CHB) patients with normal or minimally elevated serum alanine aminotransferase (ALT) levels is still debated. Animal models are crucial for studying the unanswered issue and evaluating new therapies. MicroRNA-122 (miR-122), which regulates fatty acid and cholesterol metabolism, is downregulated during hepatitis and HCC progression. The reciprocal inhibition of miR-122 with HBV highlights its role in HCC development as a tumor suppressor. By crossbreeding HBV-transgenic mice with miR-122 knockout mice, we generated a hybrid mouse model with a high incidence of HCC up to 89% and normal ALT levels before HCC. The model exhibited early-onset hepatic steatosis, progressive liver fibrosis, and impaired late-phase autophagy. Metabolomics and microarray analysis identified metabolic signatures, including dysregulation of lipid metabolism, inflammation, genomic instability, the Warburg effect, reduced TCA cycle flux, energy deficiency, and impaired free radical scavenging. Antiviral treatment reduced HCC incidence in hybrid mice by approximately 30-35% compared to untreated mice. This effect was linked to the activation of ER stress-responsive transcription factor ATF4, clearance of autophagosome cargo p62, and suppression of the CHOP-mediated apoptosis pathway. In summary, this study suggests that despite minimal ALT elevation, HBV replication can lead to liver injury. Endoplasmic reticulum stress, reduced miR-122 levels, mitochondrial and metabolic dysfunctions, blocking protective autophagy resulting in p62 accumulation, apoptosis, fibrosis, and HCC. Antiviral may improve the above-mentioned pathogenesis through HBV suppression.

Oral Zinc-Rich Oyster Supplementation Corrects Anemia in Rats.

This study investigates the impact of various zinc supplementation methods on anemia in rats induced by phenylhydrazine (PHZ) and in 5/6-nephrectomized anemic rats. We compare oral zinc sulfate (ZnSO4) supplementation, oyster Crassostrea gigas supplementation, and hard clam Meretrix lusoria supplementation on red blood cell (RBC) levels. Oral zinc-rich oyster supplementation (2.70 mg Zn (30 g oyster)/day/rat) effectively corrects anemia in both experimental groups. Rats orally fed oysters for four days exhibit similar effectiveness as those receiving a single ZnSO4 injection (0.95 mg Zn (4.18 mg ZnSO4⋅7H2O)/rat). In contrast, oral ZnSO4 supplementation (2.70 mg Zn (11.88 mg ZnSO4⋅7H2O)/day/rat) does not significantly increase RBC levels, suggesting better zinc absorption from oysters. A placebo group of anemic rats supplemented with hard clams, similar in composition to oysters but much lower in zinc, did not change RBC counts. This supports oysters' high zinc content as the key to correcting anemia. Oysters also contain high iron levels, offering a potential solution for iron-deficiency anemia while supporting bone marrow erythropoiesis. In summary, oral oyster supplementation emerges as an effective strategy to correct anemia in rats with added zinc and iron support for erythropoiesis.

HArmonized single-cell RNA-seq Cell type Assisted Deconvolution (HASCAD).

BACKGROUND: Cell composition deconvolution (CCD) is a type of bioinformatic task to estimate the cell fractions from bulk gene expression profiles, such as RNA-seq. Many CCD models were developed to perform linear regression analysis using reference gene expression signatures of distinct cell types. Reference gene expression signatures could be generated from cell-specific gene expression profiles, such as scRNA-seq. However, the batch effects and dropout events frequently observed across scRNA-seq datasets have limited the performances of CCD methods. METHODS: We developed a deep neural network (DNN) model, HASCAD, to predict the cell fractions of up to 15 immune cell types. HASCAD was trained using the bulk RNA-seq simulated from three scRNA-seq datasets that have been normalized by using a Harmony-Symphony based strategy. Mean square error and Pearson correlation coefficient were used to compare the performance of HASCAD with those of other widely used CCD methods. Two types of datasets, including a set of simulated bulk RNA-seq, and three human PBMC RNA-seq datasets, were arranged to conduct the benchmarks. RESULTS: HASCAD is useful for the investigation of the impacts of immune cell heterogeneity on the therapeutic effects of immune checkpoint inhibitors, since the target cell types include the ones known to play a role in anti-tumor immunity, such as three subtypes of CD8 T cells and three subtypes of CD4 T cells. We found that the removal of batch effects in the reference scRNA-seq datasets could benefit the task of CCD. Our benchmarks showed that HASCAD is more suitable for analyzing bulk RNA-seq data, compared with the two widely used CCD methods, CIBERSORTx and quanTIseq. We applied HASCAD to analyze the liver cancer samples of TCGA-LIHC, and found that there were significant associations of the predicted abundance of Treg and effector CD8 T cell with patients' overall survival. CONCLUSION: HASCAD could predict the cell composition of the PBMC bulk RNA-seq and classify the cell type from pure bulk RNA-seq. The model of HASCAD is available at https://github.com/holiday01/HASCAD .

Utility of Ultrasound Elastography to Evaluate Poststroke Spasticity and Therapeutic Efficacy: A Narrative Review.

Poststroke spasticity (PSS) is a common complication that affects function and daily self-care. Conservative PSS treatments include traditional rehabilitation, botulinum toxin injection, and extracorporeal shock wave therapy. Currently, the Modified Ashworth Scale and Modified Tardieu Scale are widely used tools to clinically evaluate spasticity, but the best tool for PSS assessment remained controversial. Ultrasound elastography (UE), including shear wave and strain image as the emerging method to evaluate soft tissue elasticity, became popular in clinical applications. Spastic biceps and gastrocnemius muscles were reported to be significantly stiffer compared to nonparetic muscles or healthy control using shear wave or strain elastography. More studies investigated the utility, reliability, and validity of UE in patients with PSS, but the contemporary consensus for the utility of UE in the measurement and therapeutic follow-up of PSS remained lacking. Therefore, this narrative review aimed to appraise the literature on the shear wave and strain elastography on PSS and summarize the roles of UE in assessing the therapeutic efficacy of different PSS interventions.