A proposed approach to confirm heroin administration - Regional differences in heroin purity is a major factor.

In forensic toxicology, a marker of street heroin use is urgent especially in the absence of urinary 6-monoacetylmorphine. ATM4G, the Glucuronide of Acetylated product of Thebaine compound 4 Metabolite (ATM4), arising from byproducts of street heroin synthesis has been considered as a useful marker in some European studies. However, whether ATM4G is a universal marker particularly in Southeast Asia due to 'street' heroin with high purity, it's still unclear. To investigate putative markers for different regions, ATM4G and other metabolites including the Acetylated product of Thebaine compound 3 Metabolite (ATM3) and thebaol, also originated from thebaine were detected in 552 urine samples from heroin users in Taiwan. Results were compared with that from samples collected in the UK and Germany. Only a sulfo-conjugate of ATM4, ATM4S, was detected in 28 Taiwanese users using a sensitive MS3 method whilst out of 351 samples from the UK and Germany, ATM4G was present in 91. Thebaol-glucuronide was first time detected in 118. No markers were detected in urine following herbal medicine use or poppy seed ingestion. The presence of ATM4S/ATM4G might be affected by ethnicities and heroin supplied in regions. Thebaol-glucuronide is another putative marker with ATM4G and ATM4S for street heroin use.

Autonomous capillary microfluidic devices with constant flow rate and temperature-controlled valving.

In this paper, we report on a capillary microfluidic device with constant flow rate and temperature-triggered stop valve function. It contains a PDMS channel that was grafted by a thermo-responsive polymer poly(N-isopropylacrylamide) (PNIPAm). The channel exhibits a constant capillary filling speed. By locally increasing the temperature in the channel from 20 °C to 37 °C using a microfabricated heater, a change of the surface wettability from hydrophilic to hydrophobic is obtained creating a hydrophobic stop valve. The valve can be reopened by lowering the temperature. The device is simple to fabricate and can be used as an actuatable capillary pump operating around room temperature. To understand the constant capillary filling speed, we performed contact angle measurements, in which we found slow wetting kinetics of PNIPAm-g-PDMS surfaces at temperatures below the lower critical solution temperature (LCST) of PNIPAm and fast wetting kinetics above the LCST. We interpret this as the result of the diffusive hydration process of PNIPAm below the LCST and the absence of hydration on the hydrophobic PNIPAm thin layer above the LCST.

Donkey-derived anti-CDV IgG, as a passive immunotherapy agent, can effectively increase survival rates of the experimental CDV-infected dogs.

BACKGROUND: Humoral immunity plays an important role in the prevention of canine distemper. Anti-CD virus (CDV) antibody has strong antiviral activity and is widely used in the treatment of CD. However, with the increase of CD cases, the availability of therapeutic CD antibody fell short of the clinical needs. RESULTS: The high-titer antiserum with the high-titer neutralizing activity against CDV was obtained from the donkeys (Dezhou Donkey) immunized with the inactivated CDV vaccine. The donkey anti-CDV IgG was purified from the donkey serum, which was identified to significantly inhibit the CDV replication in the cultured Vero cells and effectively reduce the clinical symptoms and increase the survival rates (75%) of CDV-infected dogs (Shih-tzu Dog), similar to that treated with the dog-derived anti-CDV IgG. These results indicate that donkey-derived IgG is a potential substitute for dog-derived IgG to treat the CD in clinic. CONCLUSIONS: Administration of donkey-derived anti-CDV IgG can ameliorate clinical symptoms and inhibit virus replication, thereby increasing the survival of CDV-infected dogs. This study opens up a new source of therapeutic antibody for CD treatment.

Inhibition of pro-inflammatory mediator expression in macrophages using wood vinegar from griffith's ash.

Macrophages are essential for host defense as they control foreign pathogens and induce acquired immune responses. Activated macrophages secrete pro-inflammatory reactive substances causing local cell and tissue inflammatory response, which helps an organism resist the invasion of foreign pathogens. Excessive or chronic inflammation can cause several diseases. Previous studies have reported that vinegar treatment decreases the levels of several inflammatory cytokines and biomarkers, including mitogen-activated protein kinases, cyclooxygenase-2, inducible nitric oxide synthase (iNOS), and nitric oxide (NO). However, the benefits of wood vinegar produced from Griffith's ash (Fraxinus formosana Hayata) in reducing inflammation have not been investigated yet. Thus, assuming that wood vinegar exerts anti-inflammatory effects in macrophages, in this study, we investigated the potential anti-inflammatory effects of the wood vinegar from Griffith's ash using a lipopolysaccharide (LPS)-induced inflammatory response model in RAW264.7 macrophages. We showed that the wood vinegar inhibited the production of iNOS, NO, and interleukin 6. In addition, we found that the wood vinegar reduced the phosphorylation levels of p38 and protein kinase C-α/δ in the LPS-stimulated RAW264.7 macrophages. Based on these results, we suggest that the produced wood vinegar can reduce inflammation in LPS-activated macrophages.

Ginsenoside M1 Induces Apoptosis and Inhibits the Migration of Human Oral Cancer Cells.

Oral squamous cell carcinoma (OSCC) accounts for 5.8% of all malignancies in Taiwan, and the incidence of OSCC is on the rise. OSCC is also a common malignancy worldwide, and the five-year survival rate remains poor. Therefore, new and effective treatments are needed to control OSCC. In the present study, we prepared ginsenoside M1 (20-O-beta-d-glucopyranosyl-20(S)-protopanaxadiol), a major deglycosylated metabolite of ginsenoside, through the biotransformation of Panax notoginseng leaves by the fungus SP-LSL-002. We investigated the anti-OSCC activity and associated mechanisms of ginsenoside M1 in vitro and in vivo. We demonstrated that ginsenoside M1 dose-dependently inhibited the viability of human OSCC SAS and OEC-M1 cells. To gain further insight into the mode of action of ginsenoside M1, we demonstrated that ginsenoside M1 increased the expression levels of Bak, Bad, and p53 and induced apoptotic DNA breaks, G1 phase arrest, PI/Annexin V double-positive staining, and caspase-3/9 activation. In addition, we demonstrated that ginsenoside M1 dose-dependently inhibited the colony formation and migration ability of SAS and OEC-M1 cells and reduced the expression of metastasis-related protein vimentin. Furthermore, oral administration or subcutaneous injection of ginsenoside M1 significantly reduced tumor growth in SAS xenograft mice. These results indicate that ginsenoside M1 can be translated into a potential therapeutic against OSCC.

Mechanistic insight into the attenuation of gouty inflammation by Taiwanese green propolis via inhibition of the NLRP3 inflammasome.

Dysregulation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including gouty arthritis. Activation of the NLRP3 inflammasome requires priming and activation signals: the priming signal controls the expression of NLRP3 and interleukin (IL)-1ß precursor (proIL-1ß), while the activation signal leads to the assembly of the NLRP3 inflammasome and to caspase-1 activation. Here, we reported the effects of the alcoholic extract of Taiwanese green propolis (TGP) on the NLRP3 inflammasome in vitro and in vivo. TGP inhibited proIL-1ß expression by reducing nuclear factor kappa B activation and reactive oxygen species (ROS) production in lipopolysaccharide-activated macrophages. Additionally, TGP also suppressed the activation signal by reducing mitochondrial damage, ROS production, lysosomal rupture, c-Jun N-terminal kinases 1/2 phosphorylation and apoptosis-associated speck-like protein oligomerization. Furthermore, we found that TGP inhibited the NLRP3 inflammasome partially via autophagy induction. In the in vivo mouse model of uric acid crystal-induced peritonitis, TGP attenuated the peritoneal recruitment of neutrophils, and the levels of IL-1ß, active caspase-1, IL-6 and monocyte chemoattractant protein-1 in lavage fluids. As a proof of principle, in this study, we purified a known compound, propolin G, from TGP and identified this compound as a potential inhibitor of the NLRP3 inflammasome. Our results indicated that TGP might be useful for ameliorating gouty inflammation via inhibition of the NLRP3 inflammasome.

Farmers' perceptions of agricultural land use changes in Nepal and their major drivers.

Historical trends show that the total area of agricultural land in Nepal has changed markedly over time, but few studies have addressed the causative drivers underlying this change. Evaluating the perceptions of farmers is an effective tool for addressing this issue because it reflects the full range of drivers associated with changes in land use. This study utilizes historical agricultural area, population, and climate data for 1910-2010, combined with a series of applied household surveys and focus group discussions to assess farmers' perceptions of these changes and identify the major drivers. The paired t-test was employed to measure differences between various groups of drivers. The total area of agricultural land in Nepal has expanded rapidly since 1910, more intensively in the southern (Tarai) and central (Hill) ecological regions of the country, and has decreased slightly near large cities in recent decades. Farmers' perceptions show that socioeconomic variables were considered to be the crucial drivers of changes in agricultural land use. The three other major drivers were grouped as: neighborhood, climate-topography, and policy drivers. In particular, farmers pointed to the high level of population growth (93.96%) as the main factor underlying the changes, and the majority of drivers are associated with this variable. Access to roads (77.36%), urbanization (33.77%), government policies (23.58%), and remittance impact (16.79%) are other notable triggering variables. The paired t-test results equating variables from different groups of drivers and ecological regions indicate varied significance (p-values range from 0.004 to 0.983). Our analysis confirms that the synergy between social and natural observations can be integrated to obtain research findings that identify scientific and social issues. The interplay between the drivers should be emphasized in developing plans for sustainable agricultural land use management.

Capsular Polysaccharide Is Involved in NLRP3 Inflammasome Activation by Klebsiella pneumoniae Serotype K1.

Klebsiella pneumoniae (strain 43816, K2 serotype) induces interleukin-1ß (IL-1ß) secretion, but neither the bacterial factor triggering the activation of these inflammasome-dependent responses nor whether they are mediated by NLRP3 or NLRC4 is known. In this study, we identified a capsular polysaccharide (K1-CPS) in K. pneumoniae (NTUH-K2044, K1 serotype), isolated from a primary pyogenic liver abscess (PLA K. pneumoniae), as the Klebsiella factor that induces IL-1ß secretion in an NLRP3-, ASC-, and caspase-1-dependent manner in macrophages. K1-CPS induced NLRP3 inflammasome activation through reactive oxygen species (ROS) generation, mitogen-activated protein kinase phosphorylation, and NF-κB activation. Inhibition of both the mitochondrial membrane permeability transition and mitochondrial ROS generation inhibited K1-CPS-mediated NLRP3 inflammasome activation. Furthermore, IL-1ß secretion in macrophages infected with PLA K. pneumoniae was shown to depend on NLRP3 but also on NLRC4 and TLR4. In macrophages infected with a K1-CPS deficiency mutant, an lipopolysaccharide (LPS) deficiency mutant, or K1-CPS and LPS double mutants, IL-1ß secretion levels were lower than those in cells infected with wild-type PLA K. pneumoniae. Our findings indicate that K1-CPS is one of the Klebsiella factors of PLA K. pneumoniae that induce IL-1ß secretion through the NLRP3 inflammasome.

Polymorphism and differentiation indices preferred for genetic analysis among cattle, goats, and sheep.

Most studies of polymorphism and differentiation are based on a single gene; few have treated differences among numerous genes. We have analyzed the parameters of conservation, polymorphism, and genetic differentiation to find the preferred index of 40 genes, with 1,614 sequences from cattle, goats, and sheep. Clustering and principal component factor (PCF) analyses revealed three PCFs: PCF1, representing polymorphism and differentiation; PCF2, representing nucleotide usage; and PCF3, representing gene flow (G ST ) and gene differentiation (δ ST ). We found significantly higher correlations (p < 0.001) with other indices for sequence conservation (r of -0.66 to -0.98), θη (0.52-0.95), and γ ST (0.54-0.96). This shows that they are preferable for estimating conservation, polymorphism, and differentiation of genes, respectively. The codon bias index was significantly different between the autosome and sex chromosomes (p < 0.01).

GERWR: Identifying the Key Pathogenicity- Associated sRNAs of Magnaporthe Oryzae Infection in Rice Based on Graph Embedding and Random Walk With Restart.

Rice blast, caused by Magnaporthe oryzae(M.oryzae), is a destructive rice disease that reduces rice yield by 10% to 30% annually. It also affects other cereal crops such as barley, wheat, rye, millet, sorghum, and maize. Small RNAs (sRNAs) play an essential regulatory role in fungus-plant interaction during the fungal invasion, but studies on pathogenic sRNAs during the fungal invasion of plants based on multi-omics data integration are rare. This paper proposes a novel approach called Graph Embedding combined with Random Walk with Restart (GERWR) to identify pathogenic sRNAs based on multi-omics data integration during M.oryzae invasion. By constructing a multi-omics network (MRMO), we identified 29 pathogenic sRNAs of rice blast fungus. Further analysis revealed that these sRNAs regulate rice genes in a many-to-many relationship, playing a significant regulatory role in the pathogenesis of rice blast disease. This paper explores the pathogenic factors of rice blast disease from the perspective of multi-omics data analysis, revealing the inherent connection between pathogenic factors of different omics. It has essential scientific significance for studying the pathogenic mechanism of rice blast fungus, the rice blast fungus-rice model system, and the pathogen-host interaction in related fields.

Diagnosis of an immunocompetent adult with acute headache and fever as Epstein-Barr virus encephalitis by mNGS of cerebrospinal fluid.

Complicated case with fever or headache of unknown origin is currently one of the main challenges in clinical diagnosis. A retrospective analysis was conducted on a 27-year-old female patient hospitalized with headache and fever, and the pathogen species were ultimately determined by metagenomic next-generation sequencing (mNGS) of cerebrospinal fluid (CSF). The culture results of CSF showed no bacterial or fungal growth. CSF cytology showed a significant increase in nucleated cells. Pathogenic index (corresponded to human gamma herpesvirus 4) of the microorganism after correcting for human background was 12846.77 with a host index (human resource) of 27822.48 by mNGS of CSF. The patient improved through antiviral treatment with ganciclovir. Epstein-Barr virus encephalitis is rare in immunocompetent adults, which can easily cause misdiagnosis and should be paid attention to. mNGS of CSF has significant advantages in the diagnosis of Epstein-Barr virus encephalitis.

Cinnamaldehyde, A Bioactive Compound from the Leaves of Cinnamomum osmophloeum Kaneh, Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice by Inhibiting the NLRP3 Inflammasome.

Inflammatory bowel disease (IBD) comprises a group of idiopathic intestinal disorders, including ulcerative colitis and Crohn's disease, significantly impacting the quality of life for affected individuals. The effective management of these conditions remains a persistent challenge. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, a complex molecular structure, regulates the production of pro-inflammatory cytokines such as interleukin-1ß. Abnormal activation of the NLRP3 inflammasome plays a pivotal role in the development of IBD, making it a compelling target for therapeutic intervention. Our research revealed that cinnamaldehyde (CA), a major bioactive compound found in the leaves of Cinnamomum osmophloeum kaneh, demonstrated a remarkable ability to alleviate colitis induced by dextran sulfate sodium (DSS) in a mouse model. This effect was attributed to CA's ability to downregulate the activation of the NLRP3 inflammasome and reduce the expression of pro-inflammatory mediators in the colon. In the mechanism study, we observed that CA inhibited the NLRP3 inflammasome in macrophages, at least partially, by enhancing the autophagic response, without reducing mitochondrial damage. These findings collectively suggest that CA holds significant potential as a therapeutic agent for enhancing the management of IBD, offering a promising avenue for further research and development.

Cinnamaldehyde inhibits the NLRP3 inflammasome by preserving mitochondrial integrity and augmenting autophagy in Shigella sonnei-infected macrophages.

BACKGROUND: Worldwide, more than 125 million people are infected with Shigella each year and develop shigellosis. In our previous study, we provided evidence that Shigella sonnei infection triggers activation of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome in macrophages. NLRP3 inflammasome is responsible for regulating the release of the proinflammatory cytokines interleukin (IL)-1ß and IL-18 through the protease caspase-1. Researchers and biotech companies have shown great interest in developing inhibitors of the NLRP3 inflammasome, recognizing it as a promising therapeutic target for several diseases. The leaves of Cinnamomum osmophloeum kaneh, an indigenous tree species in Taiwan, are rich in cinnamaldehyde (CA), a compound present in significant amounts. Our aim is to investigate how CA affects the activation of the NLRP3 inflammasome in S. sonnei-infected macrophages. METHODS: Macrophages were infected with S. sonnei, with or without CA. ELISA and Western blotting were employed to detect protein expression or phosphorylation levels. Flow cytometry was utilized to assess H2O2 production and mitochondrial damage. Fluorescent microscopy was used to detect cathepsin B activity and mitochondrial ROS production. Additionally, colony-forming units were employed to measure macrophage phagocytosis and bactericidal activity. RESULTS: CA inhibited the NLRP3 inflammasome in S. sonnei-infected macrophages by suppressing caspase-1 activation and reducing IL-1ß and IL-18 expression. CA also inhibited pyroptosis by decreasing caspase-11 and Gasdermin D activation. Mechanistically, CA reduced lysosomal damage and enhanced autophagy, while leaving mitochondrial damage, mitogen-activated protein kinase phosphorylation, and NF-κB activation unaffected. Furthermore, CA significantly boosted phagocytosis and the bactericidal activity of macrophages against S. sonnei, while reducing secretion of IL-6 and tumour necrosis factor following infection. CONCLUSION: CA shows promise as a nutraceutical for mitigating S. sonnei infection by diminishing inflammation and enhancing phagocytosis and the bactericidal activity of macrophages against S. sonnei.

The Angiotensin II Receptor Neprilysin Inhibitor LCZ696 Inhibits the NLRP3 Inflammasome By Reducing Mitochondrial Dysfunction in Macrophages and Alleviates Dextran Sulfate Sodium-induced Colitis in a Mouse Model.

The intracellular sensor protein complex known as the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome plays a crucial role in regulating inflammatory diseases by overseeing the production of interleukin (IL)-1ß and IL-18. Targeting its abnormal activation with drugs holds significant promise for inflammation treatment. This study highlights LCZ696, an angiotensin receptor-neprilysin inhibitor, as an effective suppressor of NLRP3 inflammasome activation in macrophages stimulated by ATP, nigericin, and monosodium urate. LCZ696 also reduces caspase-11 and GSDMD activation, lactate dehydrogenase release, propidium iodide uptake, and the extracellular release of NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in ATP-activated macrophages, suggesting a potential mitigation of pyroptosis. Mechanistically, LCZ696 lowers mitochondrial reactive oxygen species and preserves mitochondrial integrity. Importantly, it does not significantly impact NLRP3, proIL-1ß, inducible nitric oxide synthase, cyclooxygenase-2 expression, or NF-κB activation in lipopolysaccharide-activated macrophages. LCZ696 partially inhibits the NLRP3 inflammasome through the induction of autophagy. In an in vivo context, LCZ696 alleviates NLRP3-associated colitis in a mouse model by reducing colonic expression of IL-1ß and tumor necrosis factor-α. Collectively, these findings suggest that LCZ696 holds significant promise as a therapeutic agent for ameliorating NLRP3 inflammasome activation in various inflammatory diseases, extending beyond its established use in hypertension and heart failure treatment.

Honokiol Exhibits Anti-NLRP3 Inflammasome and Antimicrobial Properties in Neisseria gonorrhoeae-Infected Macrophages.

Purpose: The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, crucial in infectious and inflammatory diseases by regulating IL-1ß, presents a target for disease management. Neisseria gonorrhoeae causes gonorrhea in over 87 million people annually, with previous research revealing NLRP3 inflammasome activation in infected macrophages. No natural products have been reported to counteract this activation. Exploring honokiol, a phenolic compound from Chinese herbal medicine, we investigated its impact on NLRP3 inflammasome activation in N. gonorrhoeae-infected macrophages. Methods: Honokiol's impact on the protein expression of pro-inflammatory mediators was analyzed using ELISA and Western blotting. The generation of intracellular H2O2 and mitochondrial reactive oxygen species (ROS) was detected through specific fluorescent probes (CM-H2DCFDA and MitoSOX, respectively) and analyzed by flow cytometry. Mitochondrial membrane integrity was assessed using specific fluorescent probes (MitoTracker and DiOC2(3)) and analyzed by flow cytometry. Additionally, the effect of honokiol on the viability of N. gonorrhoeae was examined through an in vitro colony-forming units assay. Results: Honokiol effectively inhibits caspase-1, caspase-11 and GSDMD activation and reduces the extracellular release of IL-1ß, NLRP3, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in N. gonorrhoeae-infected macrophages. Detailed investigations have demonstrated that honokiol lowers the production of H2O2 and the phosphorylation of ERK1/2 in N. gonorrhoeae-infected macrophages. Importantly, the phosphorylation of JNK1/2 and p38 and the activation of NF-κB remain unaffected. Moreover, honokiol reduces the N. gonorrhoeae-mediated generation of reactive oxygen species within the mitochondria, preserving their integrity. Additionally, honokiol suppresses the expression of the pro-inflammatory mediator IL-6 and inducible nitric oxide synthase induced by N. gonorrhoeae independently of NLRP3. Impressively, honokiol exhibits in vitro anti-gonococcal activity against N. gonorrhoeae. Conclusion: Honokiol inhibits the NLRP3 inflammasome in N. gonorrhoeae-infected macrophages and holds great promise for further development as an active ingredient in the prevention and treatment of symptoms associated with gonorrhea.

Tracing subsequent dissemination of a cluster of gonococcal infections caused by an ST1407-related clone harbouring mosaic penA alleles in Taiwan.

OBJECTIVES: Successful clones of Neisseria gonorrhoeae multiantigen sequence typing sequence type (ST) 1407 and ST1407-related genotypes have been reported to cause cefixime and ceftriaxone treatment failure in many countries. We characterized the 47 isolates of a strain cluster of ST4378, a genotype that differs in the porB sequence by only one nucleotide from ST1407, in Taiwan during April 2006 to June 2012. METHODS: We identified 47 ST4378 isolates among our 2357 total isolates from the Gonococci-National Isolate Collection for Epidemiology. The corresponding patients' medical records were collected. The 47 isolates were further typed by multilocus sequence typing. Genes involved in ß-lactam (ponA), quinolone (gyrA and parC) and multidrug (mtrR, porB1b and pilQ) resistance were sequenced. Antimicrobial susceptibility was determined by the disc diffusion test and Etest. RESULTS: Cefixime MICs for the 47 isolates ranged from 0.016 to 0.19 mg/L and ceftriaxone MICs ranged from 0.012 to 0.094 mg/L. Forty-six of the 47 isolates had a mosaic penA allele type XXXIV and one had a new allele type XL, which appeared to be a recombinant of mosaic penA type XXXIV and non-mosaic penA type II. All of the isolates harboured nearly identical polymorphism in the ponA, gyrA, parC, mtrR, porB1b and pilQ genes. Among the 33 patients with known medical records, 25 (76%) were men who have sex with men (MSM), 3 (9%) were bisexual and 5 (15%) were heterosexual. Fourteen (42%) of the 33 patients had HIV, 8 (24%) had syphilis and 7 (21%) had both infections. CONCLUSIONS: This is the first report of a cluster of ST1407-related strains in Taiwan. ST4378 is a genotype that may develop to cause third-generation cephalosporin treatment failures. Our results showed that ST4378 strains primarily transmitted in a high-risk MSM/bisexual network. The potential of these strains to become untreatable and spread to other low-risk sexual networks should be closely monitored.

Genetic variability of PRNP in Chinese indigenous goats.

Polymorphism of the prion protein gene (PRNP) is usually associated with scrapie susceptibility or resistance. To determine the variability of PRNP in Chinese indigenous goat breeds, we isolated genomic DNA from goat blood and amplified and sequenced the coding region of the gene. We identified 10 polymorphic sites that gave rise to 28 haplotypes. Clear frequency differences were found between northern and southern breeds and confirmed by genetic distance analysis, except for the Tangshan dairy goat. Phylogeographic analysis supported the idea that northern and southern breeds might be considered separate clusters, except for the Tangshan dairy goat. The finding of significant differences in allele distribution in northern and southern goats, especially if involved in modulating resistance/susceptibility, needs to be carefully considered for the feasibility of selection plans for resistance to scrapie.

Leptin Induces MMP-1 Expression Through the RhoA/ERK1/2/NF-κB Axis in Human Intervertebral Disc Cartilage Endplate-Derived Stem Cells.

Purpose: Intervertebral disc (IVD) degeneration, associated with aging, may cause low back pain and disability, with obesity as a significant risk factor. In a prior study, we found a positive correlation between IVD degeneration and levels of matrix metalloproteinase-1 (MMP-1) and leptin. Yet, the interaction between MMP-1 and leptin in IVD degeneration is unclear. Our research seeks to explore leptin's influence on MMP-1 expression and the underlying mechanisms in human intervertebral disc cartilage endplate-derived stem cells, specifically SV40 cells. Methods: The mRNA and protein expression in leptin-stimulated SV40 cells were assessed using RT-real-time PCR and Western blotting or ELISA, respectively. We examined leptin-mediated RhoA activation through a GTP-bound RhoA pull-down assay. Furthermore, the phosphorylation levels of mitogen-activated protein kinases and AKT in leptin-stimulated SV40 cells were analyzed using Western blotting. The activation of NF-κB by leptin was investigated by assessing phosphorylation of IKKα/ß, IκBα, and NF-κB p65, along with the nuclear translocation of NF-κB p65. To understand the underlying mechanism behind leptin-mediated MMP-1 expression, we employed specific inhibitors. Results: Leptin triggered the mRNA and protein expression of MMP-1 in SV40 cells. In-depth mechanistic investigations uncovered that leptin heightened RhoA activity, promoted ERK1/2 phosphorylation, and increased NF-κB activity. However, leptin did not induce phosphorylation of JNK1/2, p38, or AKT. When we inhibited RhoA, ERK1/2, and NF-κB, it resulted in a decrease in MMP-1 expression. Conversely, inhibition of reactive oxygen species and NADPH oxidase did not yield the same outcome. Additionally, inhibiting RhoA or ERK1/2 led to a reduction in leptin-induced NF-κB activation. Moreover, inhibiting RhoA also decreased leptin-mediated ERK1/2 phosphorylation. Conclusion: These results indicated that leptin induced MMP-1 expression in SV40 cells through the RhoA/ERK1/2/NF-κB axis. This study provided the pathogenic role of leptin and suggested the potential therapeutic target for IVD degeneration.

Advances in eggshell membrane separation and solubilization technologies.

Eggshell membranes (ESM) contain 90% protein, 3% lipids, 2% sugars, and small amounts of minerals such as calcium and magnesium. Of the 90% of proteins present, 472 proteins species have been identified. ESM provide the initial mineralization platform for eggshell formation, and can be used for to produce adsorbents, cosmetics, and medical products because of their special physical structure and chemical composition. The special physical structure of the eggshell membrane, with disulfide bonds between and within the protein molecules and the cross-linking of lysine-derived and heterochain chains between the eggshell membrane, makes the membrane very difficult to dissolve, with a maximum solubility rate of only 62%. Also, the insolubility of ESM limits its development and use also any related research. Based on the physical structure and chemical composition of the eggshell membrane, this paper reviews the latest research on eggshell membrane separation and membrane protein solubilization to provide a reference for promoting the separation, dissolution, and rational development and use of the avian eggshell membrane.

Corrigendum: Advances in eggshell membrane separation and solubilization technologies.

[This corrects the article DOI: 10.3389/fvets.2023.1116126.].