Bacteriological evaluation and advanced SYBR-green multiplex real-time PCR assay for detection of minced meat adulteration.

Background: Minced meat is a valuable source of nutrients, but it is vulnerable to contamination by microorganisms commonly present in the environment. In addition, there is a risk of adulteration with cheaper meat sources, which can be harmful to consumers. Aim: It is crucial to identify meat adulteration with distinct microbiological analysis for legal, economic, religious, and public health purposes. Methods: A total of 100 minced meat samples were collected from several markets in Sharkia Governorate, Egypt. These samples were then subjected to bacteriological testing and an advanced multiplex PCR method. This method enables the detection of bovine, equine, porcine, and dog species in meat samples with just one step. Results: The adulterated samples had a higher total bacterial count and pH values compared to pure bovine meat. These differences in bacterial count and pH values were statistically significant, with p-values of 0.843 (log10) and 0.233, respectively. The frequency of Escherichia coli occurrence was 13%, and the O111 serotype was predominant in the adulterated samples. Listeria monocytogenes and Staphylococcus aureus were isolated with prevalence rates of 3% and 29%, respectively. Besides, the SYBR-green multiplex real-time PCR assay used in this study detected adulteration with dog, equine, and porcine meats in the examined samples at rates of 9%, 5%, and 4%, respectively. Conclusion: This method provides a sensitive and specific approach to detect issues related to well-being and safety.

Acid tolerance response of Salmonella during the squid storage and its amine production capacity analysis.

Salmonella exhibits a strong inducible acid tolerance response (ATR) under weak acid conditions, and can also induce high-risk strains that are highly toxic, acid resistant, and osmotic pressure resistant to aquatic products. However, the induction mechanism is not yet clear. Therefore, this study aims to simulate the slightly acidic, low-temperature, and high-protein environment during squid processing and storage. Through λRed gene knockout, exploring the effects of low-acid induction, long-term low-temperature storage, and two-component regulation on Salmonella ATR. In this study, we found the two-component system, PhoP/PhoQ and PmrA/PmrB in Salmonella regulates the amino acid metabolism system and improves bacterial acid tolerance by controlling arginine and lysine. Compared with the two indicators of total biogenic amine and diamine content, biogenic amine index and quality index were more suitable for evaluating the quality of aquatic products. The result showed that low-temperature treatment could inhibit Salmonella-induced ATR, which further explained the ATR mechanism from the amino acid metabolism.

Cyy-287, a novel pyrimidine-2,4-diamine derivative, efficiently mitigates inflammatory responses, fibrosis, and lipid synthesis in obesity-induced cardiac and hepatic dysfunction.

Background: Inflammation and metabolic disorders are important factors in the occurrence and development of obesity complications. In this study, we investigated the protective effect and underlying mechanism of a novel pyrimidine-2,4-diamine derivative, Cyy-287, on mice fed a high-fat diet (HFD). Methods: The mice were randomly separated into four groups (n ≥ 7): control (regular diet), HFD, HFD with Cyy-287 (5 mg/kg), and HFD with Cyy-287 (20 mg/kg) following HFD feeding for 10 weeks. After a 10-week administration, ALT and AST enzymes, echocardiography, immunohistochemical (IHC), Western blot (WB), Masson and Sirius Red staining were used to evaluate functional and morphological changes to the heart and liver. Microsomes from the mouse liver were extracted to quantify the total amount of CYP450 enzymes after drug treatment. Results: Cyy-287 decreased the levels of serum glucose, LDL, TC, ALT, and AST activities in HFD-treated mice. However, Cyy-287 administration increased ejection fraction (EF) and fractional shortening (FS) index of the heart. Cyy-287 inhibited histopathological changes in the heart and liver; decreased inflammatory activity; significantly diminished p38 mitogen-activated protein kinase (MAPK), the nuclear factor-kappa B (NF-κB) axis, and sterol regulatory element-binding protein-1c (SREBP-1c); and upregulated the AMP-activated protein kinase (AMPK) pathway in HFD-treated mice. Cyy-287 restored the content of hepatic CYP450 enzymes. Conclusion: These findings demonstrated that Cyy-287 protected heart and liver cells from obesity-induced damage by inhibiting inflammation, fibrosis, and lipid synthesis.

Measurement constrained emission estimates of alkylated polycyclic aromatic hydrocarbons in the Canadian Athabasca oil sands region.

Alkylated polycyclic aromatic hydrocarbons (APAH) are important contaminants of crude oil production and exhibit similar toxicity to their parent compounds. This study developed an emission inventory of APAH in a major oil sands development region of Alberta, Canada, and validated the inventory with ambient concentration measurements through dispersion modeling. The initial estimate of regional total annual emissions of 21 APAH species was 362 tonnes/year in the last decade, of which 309 and 53 tonnes/year were in particle-bound and gas-phase APAH, respectively. Fugitive dust from oil sands mining activities is the primary source of particle-bound APAH, emitting 274 tonnes/year. Other major sources of APAH include point sources (31), tailings ponds (21), anthropogenic fuel consumption from mine fleet (17), and local transportation (13). The group of species with highest emissions was C1-C4 alkylnaphthalenes (53%), followed by C1-C4 alkylphenanthrenes/anthracenes (19%), C1-C4 fluorenes (13%), and C1-C4 fluoranthenes/pyrenes and C1-C4 benz[a]anthracenes/chrysene/triphenylenes (7% each). CALPUFF dispersion modeling was performed using the APAH emissions as model input. The model-predicted annual average ambient APAH concentrations at 17 monitoring sites were 1%-52% (19% on average) lower than the measurements. Inverse dispersion modeling was then applied to adjust APAH emissions higher by 19% for each of the 21 APAH species, which resulted in a revised estimate of APAH emissions to 431 tonnes/year. With the revised emissions as model input, model bias in the predicted ambient concentration was reduced from -19% to -8%. The model results showed the highest concentrations of APAH were near tailings ponds and open mining faces and downwind areas, with total APAH concentrations being higher than 50 ng/m3.

Design, synthesis and biological evaluation of CDC20 inhibitors for treatment of triple-negative breast cancer.

The involvement of CDC20 in promoting tumor growth in different types of human cancers and it disturbs the process of cell division and impedes tumor proliferation. In this work, a novel of Apcin derivatives targeting CDC20 were designed and synthesized to evaluate for their biological activities. The inhibitory effect on the proliferation of four human tumor cell lines (MCF-7, MDA-MB-231, MDA-MB-468 and A549) was observed. Among them, compound E1 exhibited the strongest inhibitory effect on the proliferation of MDA-MB-231 cells with an IC50 value of 1.43 µM, which was significantly superior to that of Apcin. Further biological studies demonstrated that compound E1 inhibited cancer cell migration and colony formation. Furthermore, compound E1 specifically targeted CDC20 and exhibited a higher binding affinity to CDC20 compared to that of Apcin, thereby inducing cell cycle arrest in the G2/M phase of cancer cells. Moreover, it has been observed that compound E1 induces autophagy in cancer cells. In 4T1 Xenograft Models compound E1 exhibited the potential antitumor activity without obvious toxicity. These findings suggest that E1 could be regarded as a CDC20 inhibitor deserved further investigation.

An ATMND/SGI based three-way junction ratiometric fluorescent probe for rapid and sensitive detection of bleomycin.

In this work, we developed a rapid and sensitive label-free ratiometric fluorescent (FL) probe for the detection of bleomycin (BLM). The probe consists of a DNA sequence (D6) and two fluorophore groups, 2-amino-5,6,7-trimethyl-1,8-naphthalene (ATMND) and SYBR Green I (SGI). The D6 sequence could be folded into a three-way junction structure containing a C-C mismatch position in the junction pocket. The unique "Y" structure not only could entrap ATMND in the mismatch pocket with high affinity, leading to FL quenching at 408 nm, but also embed SGI in the grooves of the double-stranded portion, resulting in FL enhancement at 530 nm. In the presence of BLM-Fe(II), the "Y" structure of D6 was destroyed due to the specific cleavage of the BLM recognition site, the 5'-GT-3' site in D6. This caused the release of ATMND and SGI and thus the ratiometric signal change of FL enhancement by ATMND and FL quenching by SGI. Under optimal conditions, the ratiometric probe exhibited a linear correlation between the intensity ratio of F408/F530 and the concentration of BLM in the range of 0.5-1000 nM, with a detection limit of 0.2 nM. In addition, the probe was applied to detect BLM in human serum samples with satisfactory results, indicating its good clinical application potential.

Development of a Thermodynamically Favorable Multi-enzyme Cascade Reaction for Efficient Sustainable Production of ω-Amino Fatty Acids and α,ω-Diamines.

Invited for this issue's cover is the group of Huilei Yu at the East China University of Science and Technology. The image shows a sustainable biosynthesis route to nylon monomers from bio-based substrate α, ω-dicarboxylic acids. The Research Article itself is available at 10.1002/cssc.202301477.

Significantly Improved Cold Preservation of Rat Hind Limb Vascularized Composite Allografts Using the New PrC-210 Free Radical Scavenger.

Vascularized composite allotransplantation (VCA) represents a promising reconstructive solution primarily conducted to improve quality of life. However, tissue damage caused by cold-ischemia (CI) storage prior to transplant represents a major factor limiting widespread application. This study investigates the addition of the novel free radical scavenger PrC-210 to UW Organ Preservation Solution (UW Solution) to suppress CI-induced skeletal muscle injury in a rat hind limb amputation model. Lewis rats received systemic perfusion of UW solution +/- PrC-210 (0 mM control, 10 mM, 20 mM, 30 mM, or 40 mM), followed by bilateral transfemoral amputation. Limbs were stored in 40 mL of the same perfusate at 4 °C for 48 h. Muscle punch biopsies were taken at set times over the 48 h cold-storage period and analyzed for caspase-3,7 activity, cytochrome C levels, and qualitative histology. A single 15 s perfusion of PrC-210-containing UW Solution conferred a dose-dependent reduction in CI-induced muscle cell death over 48 h. In the presence of PrC-210, muscle cell mitochondrial cytochrome C release was equivalent to 0 h controls, with profound reductions in the caspase-3,7 apoptotic marker that correlated with limb histology. PrC-210 conferred complete prevention of ROS-induced mitochondrial lysis in vitro, as measured by cytochrome C release. We conclude that the addition of 30 mM PrC210 to UW Solution conferred the most consistent reduction in CI limb damage, and it warrants further investigation for clinical application in the VCA setting.

Enhancing CO2 capture of an aminoethylethanolamine-based non-aqueous absorbent by using tertiary amine as a proton-transfer mediator: From performance to mechanism.

Non-aqueous absorbents (NAAs) have attracted increasing attention for CO2 capture because of their great energy-saving potential. Primary diamines which can provide high CO2 absorption loading are promising candidates for formulating NAAs but suffer disadvantages in regenerability. In this study, a promising strategy that using tertiary amines (TAs) as proton-transfer mediators was proposed to enhance the regenerability of an aminoethylethanolamine (AEEA, diamine)/dimethyl sulfoxide (DMSO) (A/D) NAA. Surprisingly, some employed TAs such as N,N-diethylaminoethanol (DEEA), N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA), 3-dimethylamino-1-propanol (3DMA1P), and N,N-dimethylethanolamine (DMEA) enhanced not only the regenerability of the A/D NAA but also the CO2 absorption performance. Specifically, the CO2 absorption loading and cyclic loading were increased by about 12.7% and 15.5%-22.7%, respectively. The TA-enhanced CO2 capture mechanism was comprehensively explored via nuclear magnetic resonance technique and quantum chemical calculations. During CO2 absorption, the TA acted as an ultimate proton acceptor for AEEA-zwitterion and enabled more AEEA to form carbamate species (AEEACOO-) to store CO2, thus enhancing CO2 absorption. For CO2 desorption, the TA first provided protons directly to AEEACOO- as a proton donor; moreover, it functioned as a proton carrier and facilitated the low-energy step-wise proton transfer from protonated AEEA to AEEACOO-. Consequently, the presence of TA made it easier for AEEACOO- to obtain protons to decompose, resulting in enhanced CO2 desorption. In a word, introducing the TA as a proton-transfer mediator into the A/D NAA enhanced both the CO2 absorption performance and the regenerability, which was an efficient way to "kill two birds with one stone".

Risk assessment-based verification of the CertiPURTM limit values for toluene diamine and methylene dianiline in flexible polyurethane foam.

Flexible polyurethane foams (PUF) are used in many consumer products. PUF may contain trace levels of aromatic diamine impurities that could represent a potential health risk. The risk associated with sleeping on a PUF mattress was evaluated. Toxicity benchmarks for sensitization and non-cancer endpoints were derived from the respective points-of-departure using standard assessment factors. For the cancer endpoints, toxicity benchmarks were derived from the 25th-percentile values of animal studies. Recently published emission and migration data allowed to link exposure with the CertiPURTM voluntary quality limits of ≤5 mg.kg-1 for 2,4-toluene diamine and 4,4'-methylene dianiline in PUF. Using conservative exposure scenarios, lifetime-average daily internal doses from the combined inhalation and dermal exposures were calculated. Margins of safety for non-cancer and sensitization endpoints were >104. The theoretical excess cancer risk was ≤1.5 × 10-7. It is concluded that sleeping on a mattress that satisfies the CertiPUR limit value does not pose undue risk to consumers.

Preparation of multifunctional cellulose macromolecule blended fabrics through internal and external synergy by N1, N6-bis (ethylene oxide-2-ylmethyl) hexane-1,6-diamine.

With the diversification of people's demand for textile functions, the preparation of multifunctional fabrics is still a current research hotspot. In this study, the water-soluble epoxy compound N1, N6-bis(oxiran-2-ylmethyl) hexane-1,6-diamine (EH) was introduced into cellulose macromolecule blended fabrics (cotton/modal) by two-phase vaporization technique, resulting in excellent wrinkle, hydrophobicity, and certain UV protection effects. It could be observed by electron microscopy that EH formed a polymer film on the fiber surface. In addition, the results of EDS scans and fiber swelling rate tests showed that EH was uniformly distributed and formed a cross-linked structure in the amorphous zones inside the fibers. Compared with the control fabrics, the wrinkle recovery angle of the EH-treated fabric was increased by 39.7 %. The fabrics could reach a contact angle of 136.9°, providing excellent hydrophobic effect. In addition, the fabrics achieved certain UV protection effects (UPF of 50+). The EH-treated fabrics were less stabilized in strong acid and alkali conditions, but exhibited greater durability in other environments. In summary, the internal and external synergistic effects of EH in forming polymer films on the fibers surface and internal cross-linking structures provided a cleaner, simple, and feasible method for the preparation of multifunctional cellulose macromolecule fibers textiles.

Murine Myoblasts Exposed to SYUIQ-5 Acquire Senescence Phenotype and Differentiate into Sarcopenic-Like Myotubes, an In Vitro Study.

The musculoskeletal system is one of the most affected organs by aging that correlates well with an accumulation of senescent cells as for other multiple age-related pathologies. The molecular mechanisms underpinning muscle impairment because of senescent cells are still elusive. The availability of in vitro model of skeletal muscle senescence is limited and restricted to a small panel of phenotypic features of these senescent cells in vivo. Here, we developed a new in vitro model of senescent C2C12 mouse myoblasts that, when subjected to differentiation, the resulting myotubes showed sarcopenic features. To induce senescence, we used SYUIQ-5, a quindoline derivative molecule inhibitor of telomerase activity, leading to the expression of several senescent hallmarks in treated myoblasts. They had increased levels of p21 protein accordingly with the observed cell cycle arrest. Furthermore, they had enhanced SA-ßgalactosidase enzyme activity and phosphorylation of p53 and histone H2AX. SYUIQ-5 senescent myoblasts had impaired differentiation potential and the resulting myotubes showed increased levels of ATROGIN-1 and MURF1, ubiquitin ligases components responsible for protein degradation, and decreased mitochondria content, typical features of sarcopenic muscles. Myotubes differentiated from senescent myoblasts cultures release increased levels of MYOSTATIN that could affect skeletal muscle cell growth. Overall, our data suggest that a greater burden of senescent muscle cells could contribute to sarcopenia. This study presents a well-defined in vitro model of muscle cell senescence useful for deeper investigation in the aging research field to discover new putative therapeutic targets and senescence biomarkers associated with the aged musculoskeletal system.

The highly selective rhodol-based putrescine probe and visual sensors for on-site detection of putrescine in food spoilage.

Putrescine (Butane-1,4-diamine) has been regarded as a vital marker of spoiling protein-rich foods, especially meat and seafood. The detection of putrescine in food is considered a convenient and powerful method for evaluating the degree of spoilage of protein-rich foods. Herein, a novel rhodol-based fluorescent probe RSMA (formyl-rhodol Schiff base with methoxyaniline) was developed to detect putrescine. RSMA exhibited excellent linearity (R2 = 0.9912) in the concentration range of 0-45 µM of putrescine with a detection limit as low as 0.45 µM. Although RSMA had moderate responses to some aliphatic diamines, the selectivity of RSMA for putrescine was one of the best reported in the literature so far. Moreover, RSMA was successfully fabricated to solid-state sensors for on-site detection of putrescine in shrimp, that demonstrated its application in monitoring food spoilage.

Development and validation of cost-effective SYBR Green-based RT-qPCR and its evaluation in a sample pooling strategy for detecting SARS-CoV-2 infection in the Indonesian setting.

A low-cost SYBR Green-based RT-qPCR method to detect SARS-CoV-2 were developed and validated. Primers targeting a conserved and vital region of the N genes of SARS-CoV-2 were designed. In-silico study was performed to analyse the compatibility of the selected primer pair with Indonesian SARS-CoV-2 genome sequences available from the GISAID database. We determined the linearity of our new assay using serial dilution of SARS-CoV-2 RNA from clinical samples with known virus concentration. The assay was then evaluated using clinically relevant samples in comparison to a commercial TaqMan-based test kit. Finally, we applied the assay in sample pooling strategies for SARS-CoV-2 detection. The SYBR Green-based RT-qPCR method was successfully developed with sufficient sensitivity. There is a very low prevalence of genome variation in the selected N primer binding regions, indicating their high conservation. The validation of the assay using clinical samples demonstrated similar performance to the TaqMan method suggesting the SYBR methods is reliable. The pooling strategy by combining 5 RNA samples for SARS-CoV-2 detection using the SYBR RT-qPCR methods is feasible and provides a high diagnostic yield. However, when dealing with samples having a very low viral load, it may increase the risk of missing positive cases.

The Significance and Importance of dPCR, qPCR, and SYBR Green PCR Kit in the Detection of Numerous Diseases.

Digital PCR (dPCR) is the latest technique that has become commercially accessible for various types of research. This method uses Taq polymerase in a standard polymerase chain reaction (PCR) to amplify a target DNA fragment from a complex sample, like quantitative PCR (qPCR) and droplet digital PCR (dd- PCR). ddPCR may facilitate microRNA (miRNA) measurement, particularly in liquid biopsy, because it has been proven to be more effective and sensitive, and in this method, ddPCR can provide an unprecedented chance for deoxyribonucleic acid (DNA) methylation research because of its capability to increase sensitivity and precision over conventional PCR-based methods. qPCR has also been found to be a valuable standard technique to measure both copy DNA (cDNA) and genomic DNA (gDNA) levels, although the finding data can be significantly variable and non-reproducible without relevant validation and verification of both primers and samples. The SYBR green quantitative real-time PCR (qPCR) method has been reported as an appropriate technique for quantitative detection and species discrimination, and has been applied profitably in different experiments to determine, quantify, and discriminate species. Although both TaqMan qRT-PCR and SYBR green qRT-PCR are sensitive and rapid, the SYBR green qRT-PCR assay is easy and the TaqMan qRT-PCR assay is specific but expensive due to the probe required. This review aimed to introduce dPCR, qPCR, SYBR green PCR kit, and digital PCR, compare them, and also introduce their advantages in the detection of different diseases.

Sulfonated Polyimide Membranes Constructed by Main-Chain and Molecular-Network Engineering Strategy for Direct Methanol Fuel Cell.

Excessive swelling is one important factor that leads to high fuel permeability and limited operating concentration of methanol for proton exchange membranes. Herein, a collaborative strategy of main-chain and molecular-network engineering is applied to lower swelling ratio and improve methanol resistance for highly sulfonated polyimide. Two m-phenylenediamine monomers (4-(2,3,5,6-tetrafluoro-4-vinylphenoxy)benzene-1,3-diamine and 4,6-bis(2,3,5,6-tetrafluoro-4-vinylphenoxy)benzene-1,3-diamine) with tetrafluorostyrol groups are designed and synthesized. Two series of cross-linked sulfonated polyimides (CSPI-Ts, CSPI-Bs) are prepared from the two diamines, 4,4'-diaminostilbene-2,2'-disulfonic acid and 1,4,5,8-naphthalenetetracarboxylicdianhydride. The rigid main-chain structure is cornerstone for wet CSPI-Ts and CSPI-Bs remaining stable at elevated temperatures. The introduction of hydrophobic cross-linked network further improves their dimensional stability and methanol resistance. CSPI-Ts and CSPI-Bs show obviously improved performances containing high proton conductivity (121 ± 0.27-158 ± 0.35 S cm-1 ), low swelling ratio (9.6 ± 0.40%-16.1 ± 0.01%) and methanol permeability (4.14-7.69 × 10-7 cm2 s-1 ) at 80 °C. The direct methanol fuel cell (DMFC) is assembled from CSPI-T-10 with balanced properties, and it exhibits high maximum power density (PDmax ) of 82.3 and 72.6 mW cm-2 in 2 and 10 m methanol solution, respectively. The ratio of PDmax in 10 m methanol solution to the value in 2 m methanol solution is as high as 88%. The CSPI-T-10 is promising proton exchange membrane candidate for DMFC application.

A versatile approach for one-pot synthesis of hybridized quinolines linked to fused N-containing heterocycles in water.

Here, highly efficient one-pot protocols for the synthesis of structurally diverse fused N-containing heterocycles containing 2-chloroquinoline employing 1,1-bis(methylsulfanyl)-2-nitroethene, diamines, 2-chloroquinoline-3-carbaldehydes and dimedone/Meldrum's acid in green media in the absence of catalyst are reported. The current report proposes sustainable, simple, four-component and straightforward strategies for generating interesting N-containing heterocyclic compounds from a range of diamines and 2-chloroquinoline-3-carbaldehydes. The utilization of water as green media furnishes sustainability by preventing the usage of toxic solvent. A range of quinoline-containing aldehydes and diamines can be converted to two types of products with respect to using dimedone or Meldrum's acid via an inexpensive, one-pot and easy route.