α-synuclein-lack expression rescues methamphetamine-induced mossy fiber degeneration in dorsal hippocampal CA3.

Methamphetamine (METH) - induced cognitive impairments may be related to synaptic degeneration at mossy fiber terminals, critical for spatial memory formation in hippocampal circuits. We have previously found METH-induced neurodegeneration in the striatum by increasing the α-synuclein (α-SYN) level. However, whether and how the METH-induced mossy fiber degeneration is also blamed for the abnormal accumulation of α-SYN remains to be elucidated. Chronic METH exposure decreased mossy fiber density but upregulatedα-SYN and phosphorylated TAU (TAU-pSer396) in hippocampal CA3, associated with glial cell overactivation, axonal neuropathies, and memory impairment. Notably, the knockout of the α-SYN gene significantly alleviated the METH-induced mossy fiber degeneration and memory impairment. Meanwhile, the TAU-pSer396 accumulation and glial activation were ameliorated by α-SYN knockout. Our findings suggest an essential role of α-SYN in mediating METH-induced mossy fiber degeneration, providing promising therapeutic and prophylactic targets for METH-related neurodegenerative diseases.

Profile and clinical significance of interferon gamma-inducible protein-10 (IP-10) and its receptor in patients with hepatocellular carcinoma.

BACKGROUND: Chemokines play a vital role in tumor progression, metastasis and prognosis; however, the profile and clinical significance of gamma interferon-inducible protein-10 (IP-10) and its receptor (CXCR3) in patients with hepatocellular carcinoma (HCC) have not been well evaluated. METHODS: Liquid-phase chip technology was used to detect the serum IP-10 in 85 patients with HBV-related HCC, 50 patients with chronic hepatitis B (CHB) and 50 liver cirrhosis subjects (CS); simultaneously, the CXCR3 and Alpha fetoprotein (AFP) were determined. Additionally, their mRNA or protein expression levels in peripheral blood mononuclear cells (PBMC), liver tumor and paracancerous tissues were quantified using qRT-PCR or ELISA. Moreover, the IP-10 and CXCR3 expression was verified by the online data from Gene Expression Omnibus. Furthermore, the relationships of serum IP-10, CXCR3 and AFP levels with their overall survival rate were also analyzed. RESULTS: The levels of IP-10 and CXCR3 in HCC group were significantly higher than those in CHB and CS groups, and their mRNA of PBMC is significantly positive correlation with those in their liver tissues or HBV DNA load (P < 0.0001), respectively. The serum IP-10 and CXCR3 in HCC were significantly correlated with tumor differentiation, metastases staging and distant metastasis (P < 0.05), but not related to gender, age and tumor size (P > 0.05, except IP-10 based on age). CONCLUSIONS: The serum IP-10 (142.6 pg/mL) and CXCR3 (241.2 pg/mL) could be differential diagnostic surrogates that distinguish HCC from CS, and the lower IP-10 level may be conducive to the postoperative survival of HCC patients. Moreover, the IP-10 and CXCR3 would be related to anti-tumor immunity in HCC patients and be a potential target for treatment of HCC.

Aging Immune System and Its Correlation With Liability to Severe Lung Complications.

Aging is considered to be a decline in physical and physiological events that extensively affect the body's immunity, and is linked with deterioration in both innate and adaptive immune responses. The immune system exhibits profound age-associated variations, known as immunosenescence, comprising a significantly low production of B and T lymphocytes in bone marrow and thymus, a decreased function of mature lymphocytes in secondary lymphoid tissues, a decrease in the synthesis of fresh naïve T cells, and reduced activation of T cells. Elderly individuals face a greater risk for many diseases particularly respiratory diseases due to their poor response to immune challenges as vigorously as the young. The current review explored the aging immune system, highlight the mortality rates of severe lung complications, such as pneumonia, COVID-19, asthma, COPD, lung cancer, IPF, and acute lung injury, and their correlation with aging immunity. This study can be helpful in better understanding the pathophysiology of aging, immune responses, and developing new approaches to improve the average age of the elderly population.

Impacts of changes in intestinal flora on the metabolism of Sprague-Dawley rats.

Changes in intestinal flora affect the health and cause metabolic diseases of the host. The extent to which the impact of different changes in intestinal flora would have on the metabolism of an individual has not been reported. This study aims to investigate the effect of different changes in intestinal flora on the metabolism of Sprague-Dawley (SD) normal rats' individuals. Forty-eight SD rats were randomly divided into 6 groups (8 rats per group), which were treated with normal saline, probiotics, nonpathogenic Escherichia coli, Salmonella enteritidis, gentamicin, and magnesium sulfate, respectively. After 7 days, the ileum of each group of rats was collected and real-time polymerase chain reaction was used to analyze the composition of intestinal flora. And gas chromatography/mass spectrometry (GC/MS) was used to analyze plasma metabolic profile. The results revealed that the decrease in alanine content in the probiotics group was statistically significant, while the alanine content in the nonpathogenic Escherichia group increased significantly. Alanine, leucine, isoleucine, and serine decreased significantly in the Salmonella group. Proline and butyric acid decreased significantly in the gentamicin group. The principal component analysis showed significant differences in the Salmonella group compared with other test groups. Overall, the most significant metabolic changes were observed in SD rats in the Salmonella group, while a great similarity was observed in the probiotics, Escherichia group, and gentamicin groups compared with the normal group. Changes in intestinal flora had a certain impact on the metabolism in SD rats, especially on amino acid levels.

Improved Sample Preparation for Untargeted Metabolomics Profiling of Escherichia coli.

Metabolomics is a powerful tool that can systematically describe global changes in the metabolome of microbes, thus improving our understanding of the mechanisms of action of antibiotics and facilitating the development of next-generation antibacterial therapies. However, current sample preparation methods are not efficient or reliable for studying the effects of antibiotics on microbes. In the present study, we reported a novel sample preparation approach using cold methanol/ethylene glycol for quenching Escherichia coli, thus overcoming the loss of intracellular metabolites caused by cell membrane damage. After evaluating the extraction efficiency of several extraction methods, we employed the optimized workflow to profile the metabolome of E. coli exposed to cephalexin. In doing so, we proved the utility of the proposed approach and provided insights into the comprehensive metabolic alterations associated with antibiotic treatment. IMPORTANCE The emergence and global spread of multidrug-resistant bacteria and genes are a global problem. It is critical to understand the interactions between antibiotics and bacteria and find alternative treatments for infections when we are moving closer to a postantibiotic era. It has been demonstrated that the bacterial metabolic environment plays an important role in the modulation of antibiotic susceptibility and efficacy. In the present study, we proposed a novel metabolomic approach for intracellular metabolite profiling of E. coli, which can be used to investigate the metabolite alterations of bacteria caused by antibiotic treatment. Further understanding of antibiotic-induced perturbations of bacterial metabolism would facilitate the discovery of new therapeutic targets and pathways.

Determination of macrolides in animal tissues and egg by multi-walled carbon nanotube-based dispersive solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry.

A simple and reliable analytical method was developed for the simultaneous determination of 11 macrolides in swine, chicken, bovine, and sheep tissues (muscle, liver, kidney, and fat), as well as eggs. Samples were extracted using a mixture of acetonitrile, ethyl acetate, and methanol; dispersive solid-phase extraction purification was then performed using multi-walled carbon nanotubes as the sorbent. The analytes were separated through ultra-high performance liquid chromatography and detected by electrospray ionization on a triple quadrupole mass spectrometer. The average recoveries ranged from 83.5% to 111.4%; the corresponding intra-day and inter-day relative standard deviations were less than 13.6% and 16.4%, respectively. The limit of detection and quantification of the eggs were 0.1-0.6 and 2.0 µg/kg, respectively. For other tissues, the limits of detection and quantification were 0.1-2.0 µg/kg and 5.0 µg/kg, respectively. The proposed method was successfully employed for the analysis of real samples to demonstrate its applicability.

Occurrence of pharmaceuticals and personal care products in bottled water and assessment of the associated risks.

The occurrence of 187 pharmaceuticals and personal care products (PPCPs) was investigated in bottled water samples (35 and 33 from Chinese and foreign brands, respectively). Forty-four compounds belonging to 14 PPCP categories were detected in 56 of the 68 bottled water samples. Further, more than 35% of water samples contained at least three PPCPs, and in one particular sample, 11 different PPCPs were detected. Macrolides constituted the most prevalent PPCP category, and salbutamol, erythromycin, and azithromycin showed the highest detection frequency (17.6%). The thermal stabilities of the 187 PPCPs were determined, and the results obtained showed that only 35 out of the 187 compounds were degraded by more than 50% after boiling for 5 min. Even though the risk quotients (RQs) of detected PPCPs showed low risk levels, the RQs of 13 compounds with RQs ≥ 0.0001 were 2-4 fold higher in infants than in other life stages. Moreover, further studies are necessary to evaluate the toxicity of PPCP mixtures, the effects of PPCPs on human intestinal microbiota, and their risk of induction of drug-resistant bacteria and drug-resistant genes.

Multi-class analysis of veterinary drugs in eggs using dispersive-solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry.

A multi-residue method has been developed for the identification and quantification of 78 compounds from seven different classes of veterinary drugs in eggs. This method was based on dispersive solid phase extraction where mixed-mode cation exchange sorbent was used to combine the isolation of compounds and sample purification. The analysis was performed using ultra-high performance liquid chromatography-tandem mass spectrometry, and the chromatographic run time of one injection was 9.5 min. The mean recovery ranged from 70.5% to 119.2% and inter-day relative standard deviation was less than 17.0%. The limit of quantification ranged between 0.1 and 1 µg/kg, which was sufficient to support surveillance monitoring. Lastly, the method was successfully used to detect residues of veterinary drug in real samples. The dietary exposure risk was subsequently assessed using the results of the survey, indicating that the evaluated daily intake and percentage of acceptable daily intake were at toxicologically acceptable levels.

Association of florfenicol residues with the abundance of oxazolidinone resistance genes in livestock manures.

Livestock and poultry manures are major reservoirs of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Linezolid is a clinical medicine for humans and has never been approved for use in livestock. Interestingly, three linezolid resistance genes (cfr, optrA, and poxtA) have been detected in bacteria of animal origin, arousing public concern. This study investigated the abundance of three ARGs, cfr, optrA, and poxtA, in manures from 157 large-scale farms in China using real-time quantitative polymerase chain reaction. The residual concentrations of linezolid, florfenicol, tiamulin, and valnemulin were determined using ultra-high performance liquid chromatography-tandem mass spectrometry. A total of 140 livestock farms were tested positive for ARGs, and the positive detection rate was 89.17 %. OptrA was the most commonly detected ARG. The diversity and abundance of ARGs were significantly higher in poultry and swine manure than in bovine manure. Redundancy analysis presented a strong association between florfenicol and all the three ARGs targeted in the study, and tiamulin showed a significant correlation with optrA. Our results indicated that the residual concentration of florfenicol had a major effect on the distribution of the three ARGs in livestock manures, and extensive use of florfenicol may lead to the production of linezolid resistance genes.

Determination of carbapenems in water samples by UHPLC-MS/MS.

A rapid and reliable method for the detection of five carbapenems (biapenem, imipenem, doripenem, meropenem, and faropenem) in water was developed and validated. After acidification of water samples with acetic acid, carbapenems were isolated using a Bond Elut PPL cartridge. The target compounds were separated using ultra high performance liquid chromatography with a chromatographic run time of 5 min and detected on a triple quadrupole mass spectrometer operated in positive electrospray ionization and multiple reaction monitoring mode. Mean recoveries were in the range of 76.6-106.5%, with satisfactory intraday and interday relative standard deviations lower than 10.0 and 10.8%, respectively. The limits of detection and quantification were in the ranges of 0.05-0.2 µg/L and 0.1-0.5 µg/L, respectively, depending on the analyte. The proposed method was applied to the analysis of river samples and wastewater samples from swine farms, and no carbapenems were detected in the collected samples.

Determination of cephalexin residual level using ultra-high-performance liquid chromatography-tandem mass spectrometry: Residue depletion study in swine.

An ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed to analyze cephalexin in swine tissues, urine, and feces. Samples were extracted with 1% sulfuric acid, followed by purification using MCX cartridges. Mean recoveries were 95.4%-100.7% with inter-day relative standard deviations of <8.6%. The quantitation limit was 5 µg/kg for fat and urine, and 10 µg/kg for muscle, liver, kidney, and feces. Cephalexin residue depletion was determined using 32 healthy pigs, randomly divided into eight (seven treated and one control) groups. Treated groups were intramuscularly administered 10 mg/kg b.w. five times at 24-h intervals and euthanized 6 h and 1, 2, 3, 5, 7, and 10 days after the last injection. Cephalexin was eliminated rapidly in swine muscle, liver, fat, and feces. The highest concentrations among edible organs were detected in the kidney. Moreover, the longest elimination period of cephalexin in swine was determined in urine. These results indicated that kidney and urine were likely target matrices for cephalexin residue detection in swine.

Confirmatory Analysis of Nitroimidazoles and Hydroxy Metabolites in Honey by Dispersive-Solid Phase Extraction and Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

An ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS/MS) method was developed and validated for confirmatory analysis of four nitroimidazoles and three hydroxy metabolites in honey. Honey samples were dissolved in 2% formic acid solution and nitroimidazoles and metabolites were isolated and enriched by dispersive-solid phase extraction using mixed-mode strong cation-exchange sorbent. The determination involves separation of analytes on an UHPLC C18 column and detection by multiple reaction monitoring in positive ionization mode. The recovery of the method was ranged from 90.2 to 105.6% with inter-day relative standard deviations of less than 11.2%. The limits of detection and limits of quantification were in the ranges of 0.02⁻0.07 µg/kg and 0.05⁻0.2 µg/kg, respectively. Honey samples from the market were analyzed to demonstrate the applicability of the proposed method.

Determination of Tranquilizers in Swine Urine by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A rapid, reliable, and sensitive method was developed for the determination of ten tranquilizers in swine urine. Sample preparation was based on solid-phase extraction, which combined isolation of the compounds and sample cleanup in a single step. Separation was performed on a reversed phase C18 column by gradient elution with a chromatographic run time of seven minutes, consisting of 0.1% formic acid in water and acetonitrile as the mobile phase. Multiple reaction monitoring in positive mode was applied for data acquisition. Matrix-matched calibration was used for quantification and good linearity was obtained with coefficients of determination higher than 0.99. The average recoveries of fortified samples at concentrations between 0.05 and 10 µg/L ranged from 85% to 106% with interday relative standard deviations of less than 13% in all cases. The limits of detection and limits of quantification obtained for tranquilizers in the urine were in the ranges of 0.03⁻0.1 µg/L and 0.05⁻0.25 µg/L, respectively. The applicability of the proposed method was demonstrated by analyzing real samples; diazepam was detected at concentrations between 0.3 and 0.6 µg/L.

Dynamic Reorganization of Nucleosome Positioning in Somatic Cells after Transfer into Porcine Enucleated Oocytes.

The nucleosome, the fundamental structural unit of chromatin, is a critical regulator of gene expression. The mechanisms governing changes to nucleosome occupancy and positioning during somatic cell reprogramming remain poorly understood. We established a method for generating genome-wide nucleosome maps of porcine embryonic fibroblasts (PEF), reconstructed 1-cell embryos generated by somatic cell nuclear transfer (SCNT), and fertilized zygotes (FZ) using MNase sequencing with only 1,000 cells. We found that donor PEF chromatin, especially X chromosome, became more open after transfer into porcine oocytes and nucleosome occupancy decreased in promoters but increased in the genic regions. Nucleosome arrangements around transcriptional start sites of genes with different expression levels in somatic cells tended to become transcriptionally silent in SCNT; however, some pluripotency genes adopted transcriptionally active nucleosome arrangements. FZ and SCNT had similar characteristics, unlike PEF. This study reveals the dynamics and importance of nucleosome positioning and chromatin organization early after reprogramming.

Distribution and content of lipid droplets and mitochondria in pig parthenogenetically activated embryos after delipation.

The present study examines the effect of delipation on developmental competence and the distribution pattern of lipid droplets (LDs) and mitochondria in parthenogenetically activated (PA) pig embryos. Mature oocytes were delipated by centrifugation after partial digestion of the zonae pellucidae, subjected to parthenogenetic activation after total removal of zonae pellucidae by pronase, and then cultured in vitro up to the blastocyst stage. The contents and distributions of LDs and mitochondria in the oocytes and/or embryos were observed by staining with Oil Red O and MitoTracker Red CMXRos, respectively. The LD and mitochondrial contents were significantly reduced by the delipation process, and only smaller LDs remained in the delipated oocytes and/or embryos. Their content remained constant from the metaphase II oocyte to the blastocyst stage, but they became gradually smaller as the oocytes and/or embryos developed. The distribution pattern of the LDs in the delipated embryos changed over time and in a manner different to that seen in the controls. In the early developmental stages (1- to 4-cell stages), they were distributed peripherally and formed a ring around the nucleus. However, by the blastocyst stage, a homogeneous distribution of LDs was observed in both the inner cell mass and trophectoderm. The distribution pattern of mitochondria also changed with the development of the delipated PA embryos and again, in ways different to those seen in the controls. In the early 1- to 4-cell stages, a peripheral distribution of mitochondrial foci was observed in each blastomere. However, in blastocysts, the mitochondria were homogeneously distributed throughout the inner cell mass and trophectoderm. Although the cleavage rate at the 2- and 4-cell stages of the PA embryos was not affected by delipation (95.83 ± 2.25% vs. 97.44 ± 0.67%; 79.17 ± 4.47% vs. 84.62 ± 1.19%), it was reduced significantly in the blastocyst compared with the controls (21.67 ± 3.78% vs. 49.36 ± 1.77%). The distribution pattern of the LDs in oocytes and/or embryos at different developmental stages, and that of the mitochondria in metaphase II oocytes, was affected by delipation. The developmental competence of porcine PA embryos would appear to be affected by delipation.

Errors in the calculation of (27)Al nuclear magnetic resonance chemical shifts.

Computational chemistry is an important tool for signal assignment of 27Al nuclear magnetic resonance spectra in order to elucidate the species of aluminum(III) in aqueous solutions. The accuracy of the popular theoretical models for computing the 27Al chemical shifts was evaluated by comparing the calculated and experimental chemical shifts in more than one hundred aluminum(III) complexes. In order to differentiate the error due to the chemical shielding tensor calculation from that due to the inadequacy of the molecular geometry prediction, single-crystal X-ray diffraction determined structures were used to build the isolated molecule models for calculating the chemical shifts. The results were compared with those obtained using the calculated geometries at the B3LYP/6-31G(d) level. The isotropic chemical shielding constants computed at different levels have strong linear correlations even though the absolute values differ in tens of ppm. The root-mean-square difference between the experimental chemical shifts and the calculated values is approximately 5 ppm for the calculations based on the X-ray structures, but more than 10 ppm for the calculations based on the computed geometries. The result indicates that the popular theoretical models are adequate in calculating the chemical shifts while an accurate molecular geometry is more critical.