Biomimetic Proteoglycans Strengthen the Pericellular Matrix of Normal and Osteoarthritic Human Cartilage.

In osteoarthritis (OA), degradation of cartilage pericellular matrix (PCM), the proteoglycan-rich immediate cell microniche, is a leading event of disease initiation. This study demonstrated that biomimetic proteoglycans (BPGs) can diffuse into human cartilage from both normal and osteoarthritic donors and are preferentially localized within the PCM. Applying immunofluorescence (IF)-guided AFM nanomechanical mapping, we show that this localization of BPGs increases the PCM micromodulus of both normal and OA specimens. These results illustrate the capability of BPGs to integrate with degenerative tissues and support the translational potential of BPGs for treating human OA and other diseases associated with proteoglycan degradation.

The effect of intermittent fasting on preventing obesity-related early aging from a molecular and cellular perspective.

Obesity is a global health concern owing to its association with numerous degenerative diseases and the fact that it may lead to early aging. Various markers of aging, including telomere attrition, epigenetic alterations, altered protein homeostasis, mitochondrial dysfunction, cellular senescence, stem cell disorders, and intercellular communication, are influenced by obesity. Consequently, there is a critical need for safe and effective approaches to prevent obesity and mitigate the onset of premature aging. In recent years, intermittent fasting (IF), a dietary strategy that alternates between periods of fasting and feeding, has emerged as a promising dietary strategy that holds potential in counteracting the aging process associated with obesity. This article explores the molecular and cellular mechanisms through which IF affects obesity-related early aging. IF regulates various physiological processes and organ systems, including the liver, brain, muscles, intestines, blood, adipose tissues, endocrine system, and cardiovascular system. Moreover, IF modulates key signaling pathways such as AMP-activated protein kinase (AMPK), sirtuins, phosphatidylinositol 3-kinase (PI3K)/Akt, mammalian target of rapamycin (mTOR), and fork head box O (FOXO). By targeting these pathways, IF has the potential to attenuate aging phenotypes associated with obesity-related early aging. Overall, IF offers promising avenues for promoting healthier lifestyles and mitigating the premature aging process in individuals affected by obesity.

Intermittent Fasting: Myths, Fakes and Truth on This Dietary Regimen Approach.

Intermittent fasting (IF) has been indicated as a valuable alternative to the classical caloric restriction dietary regimen for lowering body weight and preventing obesity-related complications, such as metabolic syndrome and type II diabetes. However, is it effective? In this review article, we analyzed over 50 clinical studies in which IF, conducted by alternate day fasting (ADF) or time-restricted feeding (TRF), was compared with the caloric restriction approach. We evaluated the different roles of IF in treating and preventing human disorders such as metabolic syndrome, type II diabetes, and some types of cancer, as well as the usefulness of IF in reducing body weight and cardiovascular risk factors such as hypertension. Furthermore, we explored the cellular pathways targeted by IF to exert their beneficial effects by activating effector proteins that modulate cell functions and resistance to oxidative stress. In contrast, we investigated concerns regarding human health related to the adoption of IF dietary regimens, highlighting the profound debate surrounding weight loss regimens. We examined and compared several clinical trials to formulate an updated concept regarding IF and its therapeutic potential.

TRPA1 Covalent Ligand JT010 Modifies T Lymphocyte Activation.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non-selective cation channel involved in sensitivity to a plethora of irritating agents and endogenous mediators of oxidative stress. TRPA1 influences neuroinflammation and macrophage and lymphocyte functions, but its role is controversial in immune cells. We reported earlier a detectable, but orders-of-magnitude-lower level of Trpa1 mRNA in monocytes and lymphocytes than in sensory neurons by qRT-PCR analyses of cells from lymphoid organs of mice. Our present goals were to (a) further elucidate the expression of Trpa1 mRNA in immune cells by RNAscope in situ hybridization (ISH) and (b) test the role of TRPA1 in lymphocyte activation. RNAscope ISH confirmed that Trpa1 transcripts were detectable in CD14+ and CD4+ cells from the peritoneal cavity of mice. A selective TRPA1 agonist JT010 elevated Ca2+ levels in these cells only at high concentrations. However, a concentration-dependent inhibitory effect of JT010 was observed on T-cell receptor (TcR)-induced Ca2+ signals in CD4+ T lymphocytes, while JT010 neither modified B cell activation nor ionomycin-stimulated Ca2+ level. Based on our present and past findings, TRPA1 activation negatively modulates T lymphocyte activation, but it does not appear to be a key regulator of TcR-stimulated calcium signaling.

Covering assisted intuitionistic fuzzy bi-selection technique for data reduction and its applications.

The dimension and size of data is growing rapidly with the extensive applications of computer science and lab based engineering in daily life. Due to availability of vagueness, later uncertainty, redundancy, irrelevancy, and noise, which imposes concerns in building effective learning models. Fuzzy rough set and its extensions have been applied to deal with these issues by various data reduction approaches. However, construction of a model that can cope with all these issues simultaneously is always a challenging task. None of the studies till date has addressed all these issues simultaneously. This paper investigates a method based on the notions of intuitionistic fuzzy (IF) and rough sets to avoid these obstacles simultaneously by putting forward an interesting data reduction technique. To accomplish this task, firstly, a novel IF similarity relation is addressed. Secondly, we establish an IF rough set model on the basis of this similarity relation. Thirdly, an IF granular structure is presented by using the established similarity relation and the lower approximation. Next, the mathematical theorems are used to validate the proposed notions. Then, the importance-degree of the IF granules is employed for redundant size elimination. Further, significance-degree-preserved dimensionality reduction is discussed. Hence, simultaneous instance and feature selection for large volume of high-dimensional datasets can be performed to eliminate redundancy and irrelevancy in both dimension and size, where vagueness and later uncertainty are handled with rough and IF sets respectively, whilst noise is tackled with IF granular structure. Thereafter, a comprehensive experiment is carried out over the benchmark datasets to demonstrate the effectiveness of simultaneous feature and data point selection methods. Finally, our proposed methodology aided framework is discussed to enhance the regression performance for IC50 of Antiviral Peptides.

Benzoylaconine Protects Skeletal Muscle Against Ischemia-Reperfusion Injury Through Activation of IF1-Dependent AMPK/Nrf2 Axis.

Background: Aconitum carmichaelii (Fuzi) has been conventionally used to cure a variety of ailments, such as pain, cold sensations, and numbness of limb muscles (Bi Zheng) in China. Our prior investigations identified Benzoylaconine (BAC) as a bioactive alkaloid derived from Aconitum carmichaelii, with other studies also demonstrating its significant pharmacological potential. Purpose: This study aimed to explore the potential of BAC as a protective agent against skeletal muscle ischemia-reperfusion (I/R) injury and to elucidate the underlying mechanisms. Methods: In vivo models involved subjecting Sprague-Dawley rats to I/R through femoral artery ligation followed by reperfusion, while in vitro models utilized C2C12 cells subjected to hypoxia/reoxygenation (H/R). CCK-8 assay was used to assess cell viability. TUNEL staining and flow cytometric analysis were used to measure cell apoptosis. Biochemical assay was used to assess skeletal muscle injury and oxidative stress. Immunofluorescence and Western blot were performed to determine protein levels. Results: BAC effectively protected muscle tissue from I/R injury, enhancing cell viability (p<0.01), elevating SOD levels (p<0.05), and reducing CK (p<0.01), LDH (p<0.01), ROS (p<0.01), MDA (p<0.01), and apoptosis-related molecules in vivo and in vitro (p<0.05, p<0.01). Mechanistically, BAC increased the expression of IF1, phosphorylated AMPK, facilitated the translocation of nuclear Nrf2, and induced the expression of HO-1 (p<0.01). Notably, AMPK inhibitor Compound C significantly hindered the ability of BAC to ameliorate H/R-induced cell injury (p<0.05), oxidative stress(p<0.01), and apoptosis (p<0.05), as well as promote Nrf2 nuclear translocation (p<0.01). Moreover, silencing of IF1 with siRNA abolished BAC-induced activation of AMPK/Nrf2 axis (p<0.01). Conclusion: Our study provides novel evidence supporting the potential of BAC as a myocyte-protective agent against I/R injury, and we establish a previously unknown mechanism involving the activation of the IF1-dependent AMPK/Nrf2 axis in mediating the protective effects of BAC.

3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing.

BACKGROUND: Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity. RESULTS: We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons. CONCLUSIONS: We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.

A 5:2 intermittent fasting regimen ameliorates NASH and fibrosis and blunts HCC development via hepatic PPARα and PCK1.

The role and molecular mechanisms of intermittent fasting (IF) in non-alcoholic steatohepatitis (NASH) and its transition to hepatocellular carcinoma (HCC) are unknown. Here, we identified that an IF 5:2 regimen prevents NASH development as well as ameliorates established NASH and fibrosis without affecting total calorie intake. Furthermore, the IF 5:2 regimen blunted NASH-HCC transition when applied therapeutically. The timing, length, and number of fasting cycles as well as the type of NASH diet were critical parameters determining the benefits of fasting. Combined proteome, transcriptome, and metabolome analyses identified that peroxisome-proliferator-activated receptor alpha (PPARα) and glucocorticoid-signaling-induced PCK1 act co-operatively as hepatic executors of the fasting response. In line with this, PPARα targets and PCK1 were reduced in human NASH. Notably, only fasting initiated during the active phase of mice robustly induced glucocorticoid signaling and free-fatty-acid-induced PPARα signaling. However, hepatocyte-specific glucocorticoid receptor deletion only partially abrogated the hepatic fasting response. In contrast, the combined knockdown of Ppara and Pck1 in vivo abolished the beneficial outcomes of fasting against inflammation and fibrosis. Moreover, overexpression of Pck1 alone or together with Ppara in vivo lowered hepatic triglycerides and steatosis. Our data support the notion that the IF 5:2 regimen is a promising intervention against NASH and subsequent liver cancer.

Peptides Targeting the IF1-ATP Synthase Complex Modulate the Permeability Transition Pore in Cancer HeLa Cells.

The mitochondrial protein IF1 is upregulated in many tumors and acts as a pro-oncogenic protein through its interaction with the ATP synthase and the inhibition of apoptosis. We have recently characterized the molecular nature of the IF1-Oligomycin Sensitivity Conferring Protein (OSCP) subunit interaction; however, it remains to be determined whether this interaction could be targeted for novel anti-cancer therapeutic intervention. We generated mitochondria-targeting peptides to displace IF1 from the OSCP interaction. The use of one selective peptide led to displacement of the inhibitor IF1 from ATP synthase, as shown by immunoprecipitation. NMR spectroscopy analysis, aimed at clarifying whether these peptides were able to directly bind to the OSCP protein, identified a second peptide which showed affinity for the N-terminal region of this subunit overlapping the IF1 binding region. In situ treatment with the membrane-permeable derivatives of these peptides in HeLa cells, that are silenced for the IF1 inhibitor protein, showed significant inhibition in mitochondrial permeability transition and no effects on mitochondrial respiration. These peptides mimic the effects of the IF1 inhibitor protein in cancer HeLa cells and confirm that the IF1-OSCP interaction inhibits apoptosis. A third peptide was identified which counteracts the anti-apoptotic role of IF1, showing that OSCP is a promising target for anti-cancer therapies.

Prevalence and characterization of aminoglycoside resistance gene aph(2")-If-carrying Campylobacter jejuni.

Campylobacter jejuni is recognized as a significant foodborne pathogen, and recent studies have indicated a rising trend of aminoglycosides resistance gene aph(2″)-If among C. jejuni isolates from food-producing animals in China. However, systematic information about aph(2″)-If-positive C. jejuni from food-producing animals and other sources worldwide based on whole-genome analysis remains a knowledge gap. In this study, we aimed to analyze the worldwide distribution, genetic environment and phylogenetic tree of aph(2″)-If by utilizing Whole Genome Sequencing (WGS) data obtained, coupled with information in the GenBank database. A total of 160C. jejuni isolates in the GenBank database and 14C. jejuni isolates in our laboratory carrying aph(2″)-If gene were performed for further analysis. WGS analysis revealed the global distribution of aph(2″)-If among C. jejuni from 6 countries. Multilocus Sequence Typing(MLST) results indicated that 70 STs were involved in the dissemination of aph(2″)-If, with ST10086 being the predominant ST. Whole-genome Multilocus Sequence Typing(wg-MLST) analysis according to times, countries, and origins of C. jejuni isolation further demonstrated a close relationship between aph(2″)-If carrying C. jejuni isolates from farm and food. The findings also revealed the existence of 32 distinct types of genetic environments surrounding aph(2″)-If among these isolates. Notably, Type 30, characterized by the arrangement ISsag10-deoD-ant(9)-hp-hp-aph(2″)-If, emerged as the predominant genetic environment. In conclusion, our analysis provides the inaugural perspective on the worldwide distribution of aph(2″)-If. This resistance gene demonstrates horizontal transferability and regional diffusion in a clonal pattern. The close association observed among aph(2″)-If-positive C. jejuni strains isolated from poultry, food, and clinical environments underscores the potential for zoonotic transmission from these isolates.

Single-Cell Single-Molecule RNA-FISH Combined with Immunofluorescence and High-Speed and High-Resolution Scanning Analysis to Visualize the Reactivation of Latent HIV-1.

Latent HIV-1 reservoirs are a major obstacle to the eradication of HIV-1. Several cure strategies have been proposed to eliminate latent reservoirs. One of the key strategies involves the reactivation of latent HIV-1 from cells using latency-reversing agents. However, currently it is unclear whether any of the latency-reversing agents are able to completely reactivate HIV-1 provirus transcription in all latent cells. An understanding of the reactivation of HIV-1 provirus at single-cell single-molecule level is necessary to fully comprehend the reactivation of HIV-1 in the reservoirs. Furthermore, since reactivable viruses in the pool of latent reservoirs are rare, combining single-cell imaging techniques with the ability to visualize a large number of reactivated single cells that express both viral RNA and proteins in a pool of uninfected and non-reactivated cells will provide unprecedented information about cell-to-cell variability in reactivation. Here, we describe the single-cell single-molecule RNA-FISH (smRNA-FISH) method to visualize HIV-1 gag RNA combined with the immunofluorescence (IF) method to detect Gag protein to characterize the reactivated cells. This method allows the visualization of subcellular localization of RNA and proteins before and after reactivation and facilitates absolute quantitation of the number of transcripts per cell using FISH-quant. In addition, we describe a high-speed and high-resolution scanning (HSHRS) fluorescence microscopy imaging method to visualize rare and reactivated cells in a pool of non-reactivated cells with high efficiency.

Assessing the Socio-Economic Benefits and Costs of Insect Meal as a Fishmeal Substitute in Livestock and Aquaculture.

Sustainability targets set by the United Nations, such as Zero Hunger by 2030, encourage the search for innovative solutions to enhance food production while preserving the environment. Alternative protein sources for feed, while conventional resources like soymeal and fishmeal become more expensive and scarcer, is one of the possibilities. Studies on substituting fishmeal with insect meal show promising results in terms of animal growth and feed efficiency. This paper aims to assess the socio-economic benefits and costs of insect meal substituting fishmeal in feed and to highlight the factors influencing performance most. The study evaluates the economic value of insect-based products, waste reduction, and reduced greenhouse gas emissions as socio-economic benefits. It combines empirical data derived from laboratory trials and two case studies covering black soldier fly (Hermetia illucens) and yellow mealworm (Tenebrio molitor). Current analyses reveal negative socio-economic balances, emphasizing that reduction of operating and investment costs through upscaling and technological advancements can give a positive move, as well as factors such as current market valuations for nutrients can change significantly. Thus, a negative balance at the moment does not mean that insect rearing, and larva processing are not desirable from a long-term socio-economic perspective.

F2RL3 Regulates Epithelial-Mesenchymal Transition and Angiogenesis in Gastric Cancer through the Rap1/MAPK Signaling Pathway.

BACKGROUND: Gastric cancer (GC) is frequently diagnosed at advanced stages, when cancer cells have already metastasized. Therefore, patients with GC have a low survival rate and poor prognosis even after treatment. METHODS: We downloaded GC-related RNA sequencing (RNA-Seq) data, copy number variation (CNV) data, and clinical data for bioinformatics analysis to screen prognostic genes of GC. Single-sample gene set enrichment analysis and survival analyses were performed on the RNA-Seq data, and differential and correlation analyses were conducted on the CNV data to obtain CNV-driven differentially expressed genes (DEGs). Prognostic genes were identified through univariate Cox analyses of the CNV-driven DEGs, combined with the clinical data. F2R like thrombin or trypsin receptor 3 (F2RL3) was finally selected for verification after functional and survival analyses of the prognostic genes. RESULTS: F2RL3 expression was lower in paracancer tissue than in GC tissue, and lower in GES-1 gastric epithelial cells than in GC cells. The cell culture supernatants from F2RL3-knockdown GC cells were collected and used to culture human umbilical vein endothelial cells (HUVECs). It was observed that F2RL3 enhanced the activity, metastasis, invasion, and angiogenesis of GC cells; promoted the epithelial-mesenchymal transition (EMT) of GC cells; and impacted the Ras-associated protein 1 (Rap1)/mitogen-activated protein kinase (MAPK) pathway. To further explore the involvement of the Rap1/MAPK pathway in GC development, a pathway activator was added to GC cells with knockdown of F2RL3 expression. This pathway activator not only enhanced the activity, invasion, and migration of GC cells but also promoted the EMT and blood vessel formation. CONCLUSIONS: F2RL3 regulates the angiogenesis and EMT of GC cells through the Rap1/MAPK pathway, thus influencing the onset and progression of GC.

Diagnostic Challenges and Emerging Pathogeneses of Selected Glomerulopathies.

Recent progress in glomerular immune complex and complement-mediated diseases have refined diagnostic categories and informed mechanistic understanding of disease development in pediatric patients. Herein, we discuss selected advances in 3 categories. First, membranous nephropathy antigens are increasingly utilized to characterize disease in pediatric patients and include phospholipase A2 receptor (PLA2R), Semaphorin 3B (Sema3B), neural epidermal growth factor-like 1 (NELL1), and protocadherin FAT1, as well as the lupus membranous-associated antigens exostosin 1/2 (EXT1/2), neural cell adhesion molecule 1 (NCAM1), and transforming growth factor beta receptor 3 (TGFBR3). Second, we examine advances in techniques for paraffin and light chain immunofluorescence (IF), including the former's function as a salvage technique and their necessity for diagnosis in adolescent cases of membranous-like glomerulopathy with masked IgG kappa deposits (MGMID) and proliferative glomerulonephritis with monotypic Ig deposits (PGNMID), respectively. Finally, progress in understanding the roles of complement in pediatric glomerular disease is reviewed, with specific attention to overlapping clinical, histologic, and genetic or functional alternative complement pathway (AP) abnormalities among C3 glomerulopathy (C3G), infection-related and post-infectious GN, "atypical" post-infectious GN, immune complex mediated membranoproliferative glomerulonephritis (IC-MPGN), and atypical hemolytic uremic syndrome (aHUS).

Program Evaluation's Path to Greater Policy Relevance: Learning From Rossi's Iron Laws.

In a 1987 article, Peter R. Rossi promulgated "The Iron Law of Evaluation and Other Metallic Rules." The Metallic Laws were meant as an informal (and humorous) overstatement of the weakness of contemporary evaluations of social programs. Rossi' s underlying worry was not so much about the state of evaluation technology in the abstract, but, rather, in its inability to advance our broad understanding of social problems and what to do about them---in other words, to make evaluation policy relevant. Rossi attributed the continuing failure to develop successful "large-scale social programs" to the failure to build a strong knowledge base for this kind of "social engineering." The qualities of studies that enable such accumulated learning are variously labeled "external validity," "generalizability," "applicability," or "transferability." This Special Issue includes five papers that seek to explore and apply this understanding.

Smart City Scenario Editor for General What-If Analysis.

Due to increasing urbanization, nowadays, cities are facing challenges spanning multiple domains such as mobility, energy, environment, etc. For example, to reduce traffic congestion, energy consumption, and excessive pollution, big data gathered from legacy systems (e.g., sensors not conformant with modern standards), geographic information systems, gateways of public administrations, and Internet of Things technologies can be exploited to provide insights to assess the current status of a city. Moreover, the possibility to perform what-if analyses is fundamental to analyzing the impact of possible changes in the urban environment. The few available solutions for scenario definitions and analyses are limited to addressing a single domain and providing proprietary formats and tools, with scarce flexibility. Therefore, in this paper, we present a novel scenario model and editor integrated into the open-source Snap4City.org platform to enable several processing and what-if analyses in multiple domains. Different from state-of-the-art software, the proposed solution responds to a series of identified requirements, implements NGSIv2-compliant data models with formal descriptions of the urban context, and a scenario versioning method. Moreover, it allows us to carry out analyses on different domains, as shown with some examples. As a case study, a traffic congestion analysis is provided, confirming the validity and usefulness of the proposed solution. This work was developed in the context of CN MOST, the National Center on Sustainable Mobility in Italy, and for the Tourismo EC project.

Evaluating the metropolitan public health preparedness for pandemics using entropy-TOPSIS-IF.

Introduction: Metropolitan governance's efficacy is regularly gauged by its capability for public health preparedness, a critical component, particularly in the post-pandemic climate, as global cities reassess their mitigation abilities. This process has broader implications, curbing mortality rates and amplifying sustainability. Current methodologies for preparedness assessment lean primarily on either Subjective Evaluation-Based Assessment (SBA), predicated on experts' input on various capacity indicators, or they opt for Data-Based quantitative Assessments (DBA), chiefly utilizing public statistic data. Methods: The manuscript discusses an urgent need for integrating both SBA and DBA to adequately measure Metropolitan Public Health Pandemics Preparedness (MPHPP), thus proposing a novel entropy-TOPSIS-IF model for comprehensive evaluation of MPHPP. Within this proposed model, experts' subjective communication is transformed into quantitative data via the aggregation of fuzzy decisions, while objective data is collected from public statistics sites. Shannon's entropy and TOPSIS methods are enacted on these data sets to ascertain the optimal performer after normalization and data isotropy. Results and discussion: The core contribution of the entropy-TOPSIS-IF model lies in its assessment flexibility, making it universally applicable across various contexts, regardless of the availability of expert decisions or quantitative data. To illustrate the efficacy of the entropy-TOPSIS-IF model, a numerical application is presented, examining three Chinese metropolises through chosen criteria according to the evaluations of three experts. A sensitivity analysis is provided to further affirm the stability and robustness of the suggested MPHPP evaluation model.

Intermittent fasting along with hydroalcoholic extract of Centella-asiatica ameliorates sub-acute hypoxia-induced ischemic stroke in adult zebrafish.

Reduced blood flow (hypoxia) to the brain is thought to be the main cause of strokes because it deprives the brain of oxygen and nutrients. An increasing amount of evidence indicates that the Centella-Asiatica (HA-CA) hydroalcoholic extract has a variety of pharmacological benefits, such as antioxidant activity, neuroprotection, anti-inflammatory qualities, and angiogenesis promotion. Intermittent fasting (IF) has neurological benefits such as anti-inflammatory properties, neuroprotective effects, and the ability to enhance neuroplasticity. The current study evaluates the combined effect of IF (for 1, 6, and 12 days) along with HA-CA (daily up to 12 days) in adult zebrafish subjected to hypoxia every 5 min for 12 days followed by behavioral (novel tank and open-field tank test), biochemical (SOD, GSH-Px, and LPO), inflammatory (IL-10, IL-1ß, and TNF-α), mitochondrial enzyme activities (Complex-I, II, and IV), signaling molecules (AMPK, MAPK, GSK-3ß, Nrf2), and imaging/staining (H&E, TTC, and TEM) analysis. Results show that sub-acute hypoxia promotes the behavioral alterations, and production of radical species and alters the oxidative stress status in brain tissues of zebrafish, along with mitochondrial dysfunction, neuroinflammation, and alteration of signaling molecules. Nevertheless, HA-CA along with IF significantly ameliorates these defects in adult zebrafish as compared to their effects alone. Further, imaging analysis significantly provided evidence of infarct damage along with neuronal and mitochondrial damage which was significantly ameliorated by IF and HA-CA. The use of IF and HA-CA has been proven to enhance the physiological effects of hypoxia in all dimensions.

Investigation of Mold Flux Entrapment in Deep Oscillation Mark of Interstitial-Free Steel Shell Using Mold Simulator.

The slag entrapment defect has become a big issue for the IF steel casting process. In this study, the mechanism of mold flux entrapment in deep oscillation mark of an IF steel shell was studied by a high-temperature mold simulator. Results show that both temperature and heat flux in a copper mold become lower when mold flux B with lower melting and viscosity is used, compared with these when mold flux A with higher melting and viscosity is used. The average thickness of the slag film for mold fluxes A and B is 1.31 mm and 1.63 mm, and the consumption of them is 0.33 kg/m2 and 0.35 kg/m2, respectively. The shell for mold flux A exhibits sharper oscillation marks, while the shell for mold flux B has shallower oscillation marks. These deeper oscillation marks capture the mold flux by overflow of molten steel at the meniscus, which finally produces the slag entrapment defect in the shell.

Engineering of IF1 -susceptive bacterial F1 -ATPase.

IF1 , an inhibitor protein of mitochondrial ATP synthase, suppresses ATP hydrolytic activity of F1 . One of the unique features of IF1 is the selective inhibition in mitochondrial F1 (MF1 ); it inhibits catalysis of MF1 but does not affect F1 with bacterial origin despite high sequence homology between MF1 and bacterial F1 . Here, we aimed to engineer thermophilic Bacillus F1 (TF1 ) to confer the susceptibility to IF1 for elucidating the molecular mechanism of selective inhibition of IF1 . We first examined the IF1 -susceptibility of hybrid F1 s, composed of each subunit originating from bovine MF1 (bMF1 ) or TF1 . It was clearly shown that only the hybrid with the ß subunit of mitochondrial origin has the IF1 -susceptibility. Based on structural analysis and sequence alignment of bMF1 and TF1 , the five non-conserved residues on the C-terminus of the ß subunit were identified as the candidate responsible for the IF1 -susceptibility. These residues in TF1 were substituted with the bMF1 residues. The resultant mutant TF1 showed evident IF1 -susceptibility. Reversely, we examined the bMF1 mutant with TF1 residues at the corresponding sites, which showed significant suppression of IF1 -susceptibility, confirming the critical role of these residues. We also tested additional three substitutions with bMF1 residues in α and γ subunits that further enhanced the IF1 -susceptibility, suggesting the additive role of these residues. We discuss the molecular mechanism by which IF1 specifically recognizes F1 with mitochondrial origin, based on the present result and the structure of F1 -IF1 complex. These findings would help the development of the inhibitors targeting bacterial F1 .