Assay Research Articles

Chondrogenic potential of superficial versus cartilage layer cells of the temporomandibular joint condyle in photopolymerizable gelatin-based hydrogels.

The objectives of this study were to compare the chondrogenic potential of cells derived from different layers of Mandibular condyle cartilage and to gain further understanding of the impact of chondrogenic cues when embedded into a novel hydrogel scaffold (PGH, a polymer blend of poly (ethylene glycol), gelatin, and heparin) compared to a gelatin hydrogel scaffold (GEL). Cartilage layer cells (CLCs) and fibroblastic superficial layer cells (SLCs) were harvested from the mandibular condyle of boer goats obtained from a local abattoir. After expansion, cells were seeded into PGH and GEL hydrogels and cultured in chondrogenic media for 3 weeks. Scaffolds were harvested at 0, 1, and 3 week(s) and processed for gross appearance, histochemical, biochemical, and mechanical assays. In terms of chondrogenesis, major differences were observed between scaffold materials, but not cell types. Glycosaminoglycan (GAG) staining showed GEL scaffolds deposited GAG during the 3 week period, which was also confirmed with the biochemical testing. Moreover, GEL scaffolds had significantly higher compressive modulus and peak stress than PGH scaffolds at all time points with the largest difference seen in week 3. It can be concluded that GEL outperformed PGH in chondrogenesis. It can also be concluded that materials play a more important role in the process of chondrogenesis than the tested cell populations. Fibroblastic SLCs were shown to have similar chondrogenic potential as CLCs cells, suggesting a rich pool of progenitor cells in the superficial fibroblastic layer capable of undergoing chondrogenesis given appropriate physical and chemical cues.

Feature-Agnostic Metabolomics for Determining Effective Sub-cytotoxic Doses of Common Pesticides in Human Cells.

While classical molecular biology assays can provide a measure of cellular response to chemical challenges, they rely on a single biological phenomenon to infer a broader measure of cellular metabolic response. These methods do not always afford the necessary sensitivity to answer questions of sub-cytotoxic effects, nor do they work for all cell types. Likewise, boutique assays such as cardiomyocyte beat rate may indirectly measure cellular metabolic response, but they too, are limited to measuring a specific biological phenomenon and are often limited to a single cell type. For these reasons, toxicological researchers need new approaches to determine metabolic changes across various doses in differing cell types, especially within the low-dose regime. The data collected herein demonstrate that LC-MS/MS-based untargeted metabolomics with a feature-agnostic view of the data, combined with a suite of statistical methods including an adapted environmental threshold analysis, provides a versatile, robust, and holistic approach to directly monitoring the overall cellular metabolomic response to pesticides. When employing this method in investigating two different cell types, human cardiomyocytes and neurons, this approach revealed separate sub-cytotoxic metabolomic responses at doses of 0.1 µM and 1 µM of chlorpyrifos and carbaryl. These findings suggest that this agnostic approach to untargeted metabolomics can provide a new tool for determining effective dose by metabolomics (EDm) of chemical challenges, such as pesticides, in a direct measurement of metabolomic response that is not cell type-specific or observable using traditional assays.

Pro-Osteogenic Effect of the Nutraceutical BlastiMin Complex® in Women with Osteoporosis or Osteopenia: An Open Intervention Clinical Trial

Osteoporosis is a chronic disease that affects millions of patients worldwide and is characterized by low bone mineral density (BMD) and increased risk of fractures. Notably, natural molecules can increase BMD and exert pro-osteogenic effects. Noteworthily, the nutraceutical BlastiMin Complex® (Mivell, Italy, European Patent Application EP4205733A1) can induce differentiation of human bone marrow mesenchymal stem cells (BM-MSCs) in osteoblasts and can exert in vitro pro-osteogenic and anti-inflammatory effects. Thus, the purpose of this study was to verify the effects of BlastiMin Complex® on bone turnover markers (BTMs) and BMD in patients with senile and postmenopausal osteopenia or osteoporosis. The efficacy of BlastiMin Complex® on BTMs in serum was evaluated through biochemical assays. BMD values were analyzed by dual-energy X-ray absorptiometry (DXA) and Radiofrequency Echographic Multi Spectrometry (R.E.M.S.) techniques, and the SNPs with a role in osteoporosis development were evaluated by PCR. Clinical data obtained after 12 months of treatment showed an increase in bone turnover index, a decrease in C-reactive protein levels, and a remarkable increase in P1NP levels, indicating the induction of osteoblast proliferation and activity in the cohort of 100% female patients recruited for the study. These findings show that the nutraceutical BlastiMin Complex® could be used as an adjuvant in combination with synthetic drugs for the treatment of osteoporosis pathology.

Effects of Agro-based Adsorbents on the Biochemical Profiles of Wistar Rats Exposed to Cyanide

Background: Cyanide is a very toxic chemical that reacts with the ferric cytochrome oxidase in the mitochondrial system to form a stable complex. This complex inhibits the process of oxidative phosphorylation, thereby interrupting aerobic respiration in the organism. It is postulated that activated charcoal (AC) intercepts the ingested cyanide in the gastrointestinal tract before it is absorbed into the system. Methods: A single dose of 3 mg/kg body weight of potassium cyanide (KCN) was orally administered to the rats in each of the five groups, each consisting of 6 rats. After 15 minutes, all rats in each group were given AC from different agro-based materials. The control group (group 1) received standard commercial AC orally at 1 g/kg. group 2 received AC from plantain peels, group 3 received AC from castor oil seed shell, group 4 received AC from coconut shell and group 5 received a combination of AC from plantain peels, castor oil seed shell, and coconut shell. Blood samples were collected sequentially from rats in each group for biochemical assays using standard procedures. Results: The control group, which received KCN and standard commercial AC, exhibited the highest alanine transaminase (ALT) value (60.09±0.10 U/L) on day seven. Similarly, the highest aspartate transaminase (AST) value (196.28±0.72 U/L) was observed in the control group. Alkaline phosphate (ALP) levels followed a similar pattern. On day seven, the serum creatinine levels were 3.81±0.11 mg/dL for group 1 and 3.45±0.05 mg/dL for group 5. Subsequently, all biochemical parameters decreased after day 7, with the lowest levels recorded in rats that received AC derived from coconut shells. Conclusion: The administration of locally prepared agro-based adsorbents to Wistar rats after exposure to sublethal doses of cyanide significantly mitigated the effects of the cyanide on the liver, kidneys, and heart, as indicated by the biochemical parameters of the albino Wistar rats in the study.

Sustainable Primary Cell Banking for Topical Compound Cytotoxicity Assays: Protocol Validation on Novel Biocides and Antifungals for Optimized Burn Wound Care

Thorough biological safety testing of topical therapeutic compounds and antimicrobials is a critical prerequisite for appropriate cutaneous wound care. Increasing pathogen resistance rates to traditional antibiotics and antifungals are driving the development and registration of novel chemical entities. Although they are notably useful for animal testing reduction, the gold standard in vitro cytotoxicity assays in continuous cell lines (HaCaT keratinocytes, 3T3 fibroblasts) may be discussed from a translational relevance standpoint. The aim of this study was thus to establish and validate a sustainable primary cell banking model with a view to performing optimized in vitro cytotoxicity assay development. Primary dermal fibroblasts and adipose-derived stem cell (ASC) types were established from four infant polydactyly sources. A multi-tiered primary cell banking model was then applied to prepare highly sustainable and standardized dermal fibroblast and ASC working cell banks (WCBs), potentially allowing for millions of biological assays to be performed. The obtained cellular materials were then validated for use in cytotoxicity assays through in vitro biosafety testing of topical antiseptics (chlorhexidine, hypochlorous acid) and an antifungal compound (AR-12) of interest for optimized burn wound care. The experimental results confirmed that IC50 values were comparable between cytotoxicity assays, which were performed with cell lines and with primary cells. The results also showed that hypochlorous acid (HOCl) displayed an enhanced toxicological profile as compared to the gold standard chlorhexidine (CLX). Generally, this study demonstrated that highly sustainable primary cell sources may be established and applied for consistent topical compound biological safety assessments with enhanced translational relevance. Overall, the study underscored the safety-oriented interest of functionally benchmarking the products that are applied on burn patient wounds for the global enhancement of burn care quality.

Covalent organic framework-hybridized Ag nanoparticles as SERS substrate for highly sensitive detection of DNA bases.

Currently, research in the development of high-performance sensing platforms has increased to fulfill the needs of analysis and detection. In this study, we developed a novel type of surface-enhanced Raman scattering (SERS) chip composed of a covalent organic framework (COF)-silver nanoparticles (AgNPs) nanocomposite, and this nanocomposite was fabricated by a one-step method of ultrasonically mixing the obtained COF and AgNPs. The fabricated chip exhibited high sensitivity and repeatable SERS effects. Practical application results showed that the chip was highly sensitive and reliable and capable of detecting DNA bases (adenine) tofit an equation in the range from 0.01pM to 1nM, with an R-square of 0.97253 and a detection limit of ~0.026pM (signal-to-noise ratio (S/N) = 3). Therefore, the proposed SERS system has potential applications in biological assays.

16 Linking VHL and SETD2 in a common oncogenic pathway that converges on the mitoticspindle

Abstract Background Loss of chromosome 3p is a landmark event in clear cell renal cell carcinoma (ccRCC) that results in mono-allelic loss of VHL (von Hippel Lindau) and SETD2 (Set-domain containing 2) (and other tumor suppressors co-located on 3p). Second hits in VHL inactivate this key tumor suppressor initiating tumor progression. SETD2, a histone methyltransferase, was previously shown to have a dual function in methylating both histones and microtubules, thereby contributing to both the histone and tubulin codes. Methylation by SETD2 on microtubules occurs at the mitotic spindle and is essential for normal mitosis and cytokinesis, with loss of SETD2 acting as a strong driver of apoptosis. This raises a conundrum of how cancer cells survive early mono-allelic loss of SETD2, escaping cell death. Methods Using biochemical kinase assays and mass spectrometry we have identified SETD2 as a substrate for AURKA. In addition, we have used immunoblotting and immunofluorescence assays to probe phosphorylation on SETD2 and the impact of phosphorylation of SETD2 both on its chromatin and cytoskeleton targets. Results We have identified the mitotic kinase, Aurora kinase A (AURKA), as a regulator of SETD2. Our data uncover SETD2 as a unique substrate for phosphorylation by AURKA, with mass spectrometry identifying serine 2080 (S2080) as the site of phosphorylation on SETD2. We found phosphorylation of SETD2 by AURKA at S2080 contributes to its methyltransferase (i.e. enzymatic) activity on microtubules but does not impact chromatin methylation on H3K36 which remains unaltered. We show that VHL regulates SETD2 via AURKA, and loss of phosphorylation on SETD2 results in mitotic defects and genomic instability. Importantly, we demonstrate that inhibition of AURKA is synthetic lethal in the setting of VHL and SETD2 deficiency. Conclusions AURKA expression levels are high in VHL-null cells resulting from an inability of VHL to target AURKA for degradation and our data now highlight a direct link between VHL and SETD2, two tumor suppressors believed to independently drive RCC pathogenesis. In summary, our data reveal a tumor-specific vulnerability linked to mitotic fragility that can be precisely targeted to ultimately drive mitotic catastrophe. DOD CDMRP Funding: yes

Exosomal miR-486 derived from bone marrow mesenchymal stem cells promotes angiogenesis following cerebral ischemic injury by regulating the PTEN/Akt pathway

Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have been shown to promote angiogenesis after ischemic stroke, in which microRNAs (miRs) are believed to play an important role in exosome-mediated therapeutic effects, though the mechanism is still not clear. In this study, a series of molecular biological and cellular assays, both in vitro and in vivo, were performed to elucidate the role of exosomal miR-486 in angiogenesis following cerebral ischemic and its molecular mechanisms. Our results revealed that BMSC-Exos significantly improved neurological function and increased microvessel density in ischemic stroke rats. In vitro assays showed that BMSC-Exos promoted the proliferation, migration, and tube formation ability of oxygen–glucose deprivation/reoxygenation (OGD/R) injured rat brain microvascular endothelial cells (RBMECs). Importantly, BMSC-Exos increased the expression of miR-486 and phosphorylated protein kinase B (p-Akt) and down-regulated the protein level of phosphatase and tensin homolog (PTEN) in vivo and in vitro. Mechanistic studies demonstrated that transfection with miR-486 mimic enhanced RBMECs angiogenesis and increased p-Akt expression, while inhibited PTEN expression. On the other hand, the miR-486 inhibitor induced an opposite effect, which could be blocked by PTEN siRNA. It was thus concluded that exosomal miR-486 from BMSCs may enhance the functional recovery by promoting angiogenesis following cerebral ischemic injury, which might be related to its regulation of the PTEN/Akt pathway.

FOXC1 transcriptionally suppresses ABHD5 to inhibit the progression of renal cell carcinoma through AMPK/mTOR pathway

BackgroundIncreased activity of the transcription factor FOXC1 leads to elevated transcription of target genes, ultimately facilitating the progression of various cancer types. However, there are currently no literature reports on the role of FOXC1 in renal cell carcinoma.MethodsBy using RT-qPCR, immunohistochemistry and Western blotting, FOXC1 mRNA and protein expression was evaluated. Gain of function experiments were utilized to assess the proliferation and metastasis ability of cells. A nude mouse model was created for transplanting tumors and establishing a lung metastasis model to observe cell proliferation and spread in a living organism. Various techniques including biological analysis, CHIP assay, luciferase assay, RT-qRCR and Western blotting experiments were utilized to investigate how FOXC1 contributes to the transcription of ABHD5 on a molecular level. FOXC1 was assessed by Western blot for its impact on AMPK/mTOR signaling pathway.ResultsFOXC1 is down-regulated in RCC, causing unfavorable prognosis of patients with RCC. Further experiments showed that forced FOXC1 expression significantly restrains RCC cell growth and cell metastasis. Mechanically, FOXC1 promotes the transcription of ABHD5 to activate AMPK signal pathway to inhibit mTOR signal pathway. Finally, knockdown of ABHD5 recovered the inhibitory role of FOXC1 overexpression induced cell growth and metastasis suppression.ConclusionIn general, our study demonstrates that FOXC1 exerts its tumor suppressor role by promoting ABHD5 transcription to regulating AMPK/mTOR signal pathway. FOXC1 could serve as both a diagnostic indicator and potential treatment focus for RCC.

Sestrin2 knockout exacerbates high-fat diet induced metabolic disorders and complications in female mice

BackgroundObesity and its associated complications raise significant public concern, revealing gender disparities in the susceptibility to metabolic disorders, with females often displaying greater resistance to obesity-related metabolic disorder than males. Sestrin2 is a crucial protein involved in metabolism and energy balance. This study seeks to explore whether Sesn2 knockout (KO) exacerbates high-fat diet (HFD) induced obesity in female mice.MethodsFemale mice with wild-type (WT) and Sesn2 KO were subjected to a 12-week regimen of normal diet or HFD. Using a Body Composition Analyzer, body composition was gauged. Biochemical assays encompassed glucose, lipid, and liver function measurements, alongside 24-hour urine albumin excretion. Echocardiographic evaluation assessed cardiac function. Histopathological analysis of key metabolic tissues (liver, kidney, and heart tissues) were conducted. Western blotting or qRT-PCR evaluated key proteins and genes linked to inflammation, mitochondrial, and lipid metabolism in adipose tissues.ResultsIn comparison to mice fed a regular diet, those on a HFD exhibited significant increases in body weight and fat mass. Notably, Sesn2 KO further aggravated obesity, showcasing the most pronounced metabolic anomalies: elevated body weight, fat mass, impaired glucose tolerance, and insulin sensitivity, alongside heightened levels of free fatty acids and triglycerides. Additionally, KO-HFD mice displayed exacerbated multi-tissue impairments, including elevated hepatic enzymes, increased urinary albumin excretion, compromised cardiac function, and accumulation of lipids in the liver, kidney, and heart. Moreover, adipose tissue showcased altered lipid dynamics and function, characterized by enhanced triglyceride breakdown and modified adipokine levels. Browning was diminished, along with decreased Pgc1α and Sirt1 in KO-HFD mice.ConclusionSesn2 KO exacerbates HFD-induced obesity and metabolic disorders in female mice. These findings underscore Sestrin2’s novel role as a regulator of obesity in female mice.

Structure and assembly of the human IL-12 signaling complex.

Interleukin (IL)-12 is a heterodimeric pro-inflammatory cytokine. Our cryoelectron microscopy structure determination of human IL-12 in complex with IL-12Rβ1 and IL-12Rβ2 at a resolution of 3.75Å reveals that IL-12Rβ2 primarily interacts with the IL-12p35 subunit via its N-terminal Ig-like domain, while IL-12Rβ1 binds to the p40 subunit with its N-terminal fibronectin III domain. This binding mode of IL-12 with its receptors is similar to that of IL-23 but shows notable differences with other cytokines. Through structural information and biochemical assays, we identified Y62, Y189, and K192 as key residues in IL-12p35, which bind to IL-12Rβ2 with high affinity and mediate IL-12 signal transduction. Furthermore, structural comparisons reveal two distinctive conformational states and structural plasticity of the heterodimeric interface in IL-12. As a result, our study advances our understanding of IL-12 signal initiation and opens up new opportunities for the engineering and therapeutic targeting of IL-12.

Boosting of retinol activity using novel lecithin: Retinol acyltransferase inhibitors.

Lecithin:retinol acyltransferase (LRAT) is the main enzyme catalysing the esterification of retinol to retinyl esters and, hence, is of central importance for retinol homeostasis. As retinol, by its metabolite retinoic acid, stimulates fibroblasts to synthesize collagen fibres and inhibits collagen-degrading enzymes, the inhibition of LRAT presents an intriguing strategy for anti-ageing ingredients by increasing the available retinol in the skin. Here, we synthesized several derivatives mimicking natural lecithin substrates as potential LRAT inhibitors. By exploring various chemical modifications of the core scaffold consisting of a central amino acid and an N-terminal acylsulfone, we explored 10 different compounds in a biochemical assay, resulting in two compounds with IC50 values of 21.1 and 32.7 μM (compounds 1 and 2), along with a simpler arginine derivative with comparative inhibitory potency. Supported by computational methods, we investigated their structure-activity relationship, resulting in the identification of several structural features associated with high inhibition of LRAT. Ultimately, we conducted an exvivo study with human skin, demonstrating an increase of collagen III associated with a reduction of the skin ageing process. In conclusion, the reported compounds offer a promising approach to boost retinol abundance in human skin and might present a new generation of anti-ageing ingredients for cosmetic application.

Marine microbial exopolysaccharide route synthesis of Ag and Pd metal nanoparticles: A possible anticancer, and antioxidant applications

The present research aimed develop an innovative and efficient method for fabricating silver and palladium nanoparticles (NPs)by using exopolysaccharide (EPS) from the seaweed-associated endo-symbiotic bacteria Exiguobacterium profundum (OP919469). Through, the application is a combined precipitation approach, EPS acts as a reducing and stabilising agent for the production of NPs. The major functional groups of NPs such as C–H, N–H, C–C, and C–O stretching’s were detected. The structural morphology of NPs showed a mixture of needle, spherical, and triangle-shaped with size between 10 and 50 nm. The purity of EPS was confirmed by high-performance liquid chromatography which revealed 99.81% purity. The NPs revealed strong anti-cancer and antioxidant properties. In addition, the NPs have been shown to dose-dependent toxicity with reduced hatching, survival rate, and abnormalities in zebrafish. The biochemical assay revealed that the EPS loaded NPsdid not induce oxidative stress up to 120 hpf in contrast to hydrogen peroxide which had a significant effect (P < 0.0001) on glutathione-S-transferase, catalase (P < 0.0017), and superoxide dismutase (P < 0.0001). Overall, our finding indicates that the molecular and functional characteristics of EPS and EPS loaded NPs represent future potential applications in the drug delivery and healthcare sectors.

Extracellular matrix regulation of cell spheroid invasion in a 3D bioprinted solid tumor-on-a-chip

Tumor organoids and tumors-on-chips can be built by placing patient-derived cells within an extracellular matrix (ECM) for personalized medicine. The ECM influences the tumor response, and understanding the ECM-tumor relationship is important before translating tumor-on-chips into clinics. In this work, we tuned the physical and structural characteristics of ECM in a bioprinted soft-tissue sarcoma microtissue. We formed 3D spheroids at a controlled size and encapsulated them into our gelatin methacryloyl (GelMA)-based bioink to make perfusable hydrogel-based microfluidic chips. We then demonstrated the scalability and customization flexibility of our hydrogel-based chip via engineering tools. A multiscale physical and structural data analysis suggested a relationship between cell invasion response and bioink characteristics. Tumor cell invasive behavior and focal adhesion properties were observed in response to varying polymer network densities of the GelMA-based bioink. Immunostaining assays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) helped assess the bioactivity of the microtissue and measure the cell invasion. The RT-qPCR results showed higher expressions of HIF-1α, CD44, and MMP2 genes in a lower polymer density, highlighting the correlation between bioink structural porosity, ECM stiffness, and tumor spheroid response. In conclusion, this work is the first step in modeling STS tumor invasiveness in hydrogel-based microfluidic chips, and our tunable bioink may help reduce the variability of current tumor-on-chips. Statement of SignificanceWe optimized an engineering protocol for making tumor spheroids at a controlled size, embedding spheroids into a gelatin-based matrix, and constructing a perfusable microfluidic device. A higher tumor invasion was observed in a low-stiffness matrix than a high-stiffness matrix. The physical characterizations revealed how the stiffness is controlled by the density of polymer chain networks and porosity. The biological assays revealed how the structural properties of the gelatin matrix and hypoxia in tumor progression impact cell invasion. The cell spheroids' responses underscore the importance of replicating physical and structural properties to mimic tumor response. This work can contribute to personalized medicine by making more effective, tailored cancer models.

Evening primrose and its compounds of 1-Oxohederagenin and remangilone C ameliorate bleomycin-induced pulmonary fibrosis by regulating β-catenin signaling

BackgroundPulmonary fibrosis (PF) is a progressive and severe respiratory disease for which there is still a lack of satisfactory treatment methods other than lung transplantation. Evening primrose (EP) is widely used in Chinese folk medicinal herbs, especially for the treatment of lung-related diseases. However, the protective effect of evening primrose against PF has yet to be reported. PurposeThis study explores the pharmacological effect of EP and its possible active components against PF from the perspectives of lung function, histopathological staining, and molecular biology assays. MethodsEstablishing a rat pulmonary fibrosis model using bleomycin to detect lung function, pathological changes, and collagen deposition. TGF-β1 was used to establish an in vitro model of PF in BEAS-2B cells, and the active ingredients in evening primrose were screened. Then, the therapeutic effects of 1-Oxohederagenin (C1) and remangilone C (C2) derived from EP were observed in an in vivo model of bleomycin-induced PF, and the differentially expressed genes between the C1 and C2 treatment groups and the model group were screened with transcriptome sequencing. Finally, TGF-β1-induced damage to HFL1 cell was used to explore the specific mechanisms by which C1 and C2 alleviate PF and the involvement of β-catenin signaling. ResultsEvening primrose extract showed some ameliorative effects on bleomycin-induced PF in rats, manifested as reduced pathological damage and reduced collagen deposition. The chemical components of C1 and C2 potently ameliorated BLM-induced PF in animals and effectively inhibited fibroblast activation by interfering with β-catenin signaling. ConclusionEvening primrose extract has certain ameliorative effects on PF. In addation, C1 and C2 might be related with the suppression of fibroblast activation by inhibiting β-catenin signaling.

Phosphorylation of PFKL regulates metabolic reprogramming in macrophages following pattern recognition receptor activation

Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1β than PfklS775A/S775A after LPS treatment. In an in vivo inflammation model, PfklS775A/S775A mice show reduced levels of MCP-1 and IL-1β. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis.

Super-relaxed myosins contribute to respiratory muscle hibernation in mechanically ventilated patients.

Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients (N = 54) to those of non-ICU patients undergoing thoracic surgery (N = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.

Surface Charge-Modulated Toxicity of Cysteine-Stabilized Silver Nanoparticles

The toxicity of silver nanoparticles (AgNPs) depends on their physicochemical properties. The ongoing research aims to develop effective methods for modifying AgNPs using molecules that enable control over the processes induced by nanoparticles in both normal and cancerous cells. Application of amino acid-stabilized nanoparticles appears promising, exhibiting tunable electrokinetic properties. Therefore, this study focused on determining the influence of the surface charge of cysteine (CYS)-stabilized AgNPs on their toxicity towards human normal B (COLO-720L) and T (HUT-78) lymphocyte cell lines. CYS-AgNPs were synthesized via the chemical reduction. Transmission electron microcopy (TEM) imaging revealed that they exhibited a quasi-spherical shape with an average size of 18 ± 3 nm. CYS-AgNPs remained stable under mild acidic (pH 4.0) and alkaline (7.4 and 9.0) conditions, with an isoelectric point observed at pH 5.1. Following a 24 h treatment of lymphocytes with CYS-AgNPs, concentration-dependent alterations in cell morphology were observed. Positively charged CYS-AgNPs notably decreased lymphocyte viability. Furthermore, they exhibited grater genotoxicity and more pronounced disruption of biological membranes compared to negatively charged CYZ-AgNPs. Despite both types of AgNPs interacting similarly with fetal bovine serum (FBS) and showing comparable profiles of silver ion release, the biological assays consistently revealed that the positively charged CYS-AgNPs exerted stronger effects at all investigated cellular levels. Although both types of CYS-AgNPs have the same chemical structure in their stabilizing layers, the pH-induced alterations in their surface charge significantly affect their biological activity.

Effects of Different Drying Methods on the Structural Characteristics and Multiple Bioactivities of Rosa roxburghii Tratt Fruit Polysaccharides

Drying conditions significantly impact the compositions and microstructures of polysaccharides, leading to various effects on their chemical characteristics and bioactivities. The objective of this study was to investigate how different industrial drying techniques, i.e., hot air drying, infrared drying, microwave vacuum drying, and freeze drying, affect the structural properties and biological activities of polysaccharides extracted from Rosa roxburghii Tratt fruit (RRTP). Results revealed that these drying methods significantly altered the extraction yield, molecular weights, monosaccharide ratios, contents of uronic acid and total sugars, gelling properties, particle sizes, thermal stability, and microstructures of RRTPs. However, the monosaccharide composition and functional groups of polysaccharides remained consistent across the different drying techniques. Biological activity assays demonstrated that RRTPs, particularly those processed through microwave vacuum drying (MVD-RRTP), exhibited excellent anti-linoleic acid oxidation, robust anti-glycosylation effects, and significant α-glucosidase inhibition in vitro. The outcomes of this research demonstrate that microwave vacuum drying serves as an effective pre-extraction drying method for RRTPs, enhancing their biological activities. This technique is particularly advantageous for preparing RRTPs intended for use in functional foods and pharmaceuticals, optimizing their health-promoting properties for industrial applications.

Transcriptional RNA sequencing reveals the molecular mechanism of dielectric barrier discharge cold plasma against postharvest pathogen Alternaria alternata

Alternaria alternata (A. alternata), a pervasive pathogen, infects fruits and vegetables, posing a significant contamination threat. Dielectric barrier discharge cold plasma (DBD-CP), a novel physical treatment technology, effectively prevents and controls postharvest diseases. This study investigated CP treatment's antifungal mechanism on A. alternata. Biochemical assays, laser confocal microscopy, flow cytometry analysis, and fluorescence staining demonstrated that CP treatment reduces spore germination, inhibits hyphal growth, disrupts cell membrane integrity and permeability, induces intracellular accumulation of reactive oxygen species, and interferes with energy metabolism. Specifically, following 100-kV CP treatment, hyphal growth was entirely inhibited within 24 h, and the spore germination rate decreased to only 18.48% of that of the control group. Furthermore, CP treatment significantly reduced the quantity of A. alternata inoculated on dried goji berries, with 80-kV CP treatment resulting in a 48.2% reduction. According to transcriptomic analysis, CP treatment resulted in 2954 differentially expressed genes. Among these genes, CP treatment downregulated those associated with membrane composition and functionality, DNA replication and repair, oxidative stress, and energy metabolism pathways. These findings highlight CP as a promising alternative technique for controlling postharvest fungal diseases.