Depletion Studies Research Articles

Faking at work, struggling to be healthy at home: A model of surface acting and its relation with unhealthy eating and physical activity.

Performing emotional labor impairs mood as well as regulatory control of employees, and we compare these mechanisms to explain critical health-related behaviors: eating and exercise. Two studies examine the relationship of surface acting at work with unhealthy eating and physical activity at home as mediated by negative and positive affect. Emotion regulation (ER) self-efficacy is tested as a moderator of the indirect relationships. Also, the relative strength of the affect mechanisms and a depletion mechanism are explored. Study 1 utilized a time-lagged between-person design to explore these relationships among 287 customer service workers. Results show that surface acting has an indirect link with unhealthy eating via negative affect and an indirect link with physical activity via positive affect. ER self-efficacy moderates the path from negative affect to unhealthy eating, thereby weakening the indirect effect of surface acting on unhealthy eating via negative affect. Study 2 utilized a 5-week diary study of 469 teachers to replicate Study 1 at the within-person level. Results show that surface acting has indirect effects on unhealthy eating and physical activity via negative affect at the week level. Also, surface acting has an indirect effect on physical activity via positive affect at the week level. Again, ER self-efficacy cross-moderates (weakens) the indirect link between surface acting and unhealthy eating via negative affect. The pairwise comparisons of the indirect effects reveal no differences among the affect mediators (Studies 1 and 2); however, positive affect may account for more of the effect of surface acting on physical activity than depletion (Study 2). (PsycInfo Database Record (c) 2022 APA, all rights reserved).

CD4 T-cell depletion prevents Lassa fever associated hearing loss in the mouse model.

Lassa virus (LASV) is the causative agent of Lassa fever (LF), which presents as a lethal hemorrhagic disease in severe cases. LASV-induced hearing loss in survivors is a huge socioeconomic burden, however, the mechanism(s) leading to hearing loss is unknown. In this study, we evaluate in a mouse LF model the auditory function using auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) to determine the mechanisms underlying LASV-induced hearing loss. In the process, we pioneered measures of ABR and DPOAE tests in rodents in biosafety level 4 (BSL-4) facilities. Our T cell depletion studies demonstrated that CD4 T-cells play an important role in LASV-induced hearing loss, while CD8 T-cells are critical for the pathogenicity in the acute phase of LASV infection. Results presented in this study may help to develop future countermeasures against acute disease and LASV-induced hearing loss.

Batf3-dependent regulation of the antifungal inflammatory response after pulmonary Aspergillus fumigatus challenge

Abstract Aspergillus fumigatus is a ubiquitous fungus that causes invasive aspergillosis in immunocompromised individuals. The innate immune system is critical in the maintenance of host resistance against A. fumigatus. While it is well established that monocyte-derived dendritic cells (moDC) are critical in maintaining host resistance against A. fumigatus, the role of other dendritic cells populations has not been thoroughly explored. Here we demonstrate that Batf3-dependent cells, likely classical type 1 dendritic cells (cDC1s), are critical in preventing invasive aspergillosis. Surprisingly, Batf3(−/−) mice display an elevated inflammatory response against A. fumigatus, particularly IL-17A/F. We show that elevated concentrations of IL-17A/F in the lungs of Batf3(−/−) mice following A. fumigatus infection perpetuates the secretion of various inflammatory lung cytokines in vivo. Neutralization of both IL-17A and IL-17F in Batf3(−/−) mice prevent excessive cytokine secretion and mortality due to A. fumigatus challenge of Batf3(−/−) mice. Following A. fumigatus challenge regulatory T cells are known to limit inflammation. Our preliminary antibody-mediated depletion studies demonstrate that CD4+ CD25+ T cells may be involved in preventing excessive IL-17A/F release in immunocompetent mice. In this study we have identified a critical role for Batf3-dependent cells in limiting A. fumigatus induced inflammatory and mortality due to IL-17 driven lung pathology. Supported by NIH: [R01 AI139133-01A1]

Long-term sterile immunity induced by an adjuvant-containing live-attenuated AIDS virus

Abstract Antigen 85B (Ag85B) is one of the most dominant proteins secreted from most mycobacterial species, and it induces Th1-type immune responses as an adjuvant. In the present study, a new strategy to improve live-attenuated SHIV was examined by genetically engineered virus that co-expresses an immunostimulatory molecule Ag85B such as an adjuvant. We genetically constructed a live nef-deleted simian-human immunodeficiency virus to express the adjuvant molecule Ag85B (SHIV-Ag85B) and assessed vaccine effects in cynomolgus macaques. The SHIV-Ag85B could not be detected 4 weeks after injection in cynomolgus macaques, and strong SHIV-specific T cell responses were induced in these macaques. Moreover, most of macaques inoculated with SHIV-Ag85B showed protective effects against the intravenous challenge of pathogenic SHIV89.6P, and viral RNAs remained undetectable for more than 200 weeks. Remarkably, eradication of SHIV89.6P was confirmed by an adoptive transfer experiment and CD8+ cell depletion study. In these macaques, SHIV antigen-specific CD8+ T cell responses with polyfunctionality were rapidly induced in the acute phase of SHIV89.6P challenge. The results suggest that SHIV-Ag85B elicited strong sterile immune responses against pathogenic SHIV and that it may lead to the development of a live vaccine and cure for AIDS virus infection. Supported by Health Science Research Grants from the Ministry of Health, Labor and Welfare of Japan, Ministry of Education, Culture, Sports, Science and Technology, Japan, Japan Agency for Medical Research and Development, AMED (19ak0101047h0004, 19fk0410025h0001, 19ak0101068h0003 and 19fk0410011h0702).

Addition of interleukin-2 overcomes resistance to neoadjuvant CTLA4 and PD1 blockade in ex vivo patient tumors.

Neoadjuvant immunotherapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) + anti-programmed cell death protein 1 (PD1) monoclonal antibodies has demonstrated remarkable pathological responses and relapse-free survival in ~80% of patients with clinically detectable stage III melanoma. However, about 20% of the treated patients do not respond. In pretreatment biopsies of patients with melanoma, we found that resistance to neoadjuvant CTLA4 + PD1 blockade was associated with a low CD4/interleukin-2 (IL-2) gene signature. Ex vivo, addition of IL-2 to CTLA4 + PD1 blockade induced T cell activation and deep immunological responses in anti-CTLA4 + anti-PD1-resistant human tumor specimens. In the 4T1.2 breast cancer mouse model of neoadjuvant immunotherapy, triple combination of anti-CTLA4 + anti-PD1 + IL-2 cured almost twice as many mice as compared with dual checkpoint inhibitor therapy. This improved efficacy was due to the expansion of tumor-specific CD8+ T cells and improved proinflammatory cytokine polyfunctionality of both CD4+ and CD8+ T effector cells and regulatory T cells. Depletion studies suggested that CD4+ T cells were critical for priming of CD8+ T cell immunity against 4T1.2 and helped in the expansion of tumor-specific CD8+ T cells early after neoadjuvant triple immunotherapy. Our results suggest that the addition of IL-2 can overcome resistance to neoadjuvant anti-CTLA4 + anti-PD1, providing the rationale for testing this combination as a neoadjuvant therapy in patients with early-stage cancer.

Evidence for Constitutive Microbiota-Dependent Short-Term Control of Food Intake in Mice: Is There a Link with Inflammation, Oxidative Stress, Endotoxemia, and GLP-1?

Aims: Although prebiotics, probiotics, and fecal transplantation can alter the sensation of hunger and/or feeding behavior, the role of the constitutive gut microbiota in the short-term regulation of food intake during normal physiology is still unclear. Results: An antibiotic-induced microbiota depletion study was designed to compare feeding behavior in conventional and microbiota-depleted mice. Tissues were sampled to characterize the time profile of microbiota-derived signals in mice during consumption of either standard or high-fat food for 1 h. Pharmacological and genetic tools were used to evaluate the contribution of postprandial endotoxemia and inflammatory responses in the short-term regulation of food intake. We observed constitutive microbial and macronutrient-dependent control of food intake at the time scale of a meal; that is, within 1 h of food introduction. Specifically, microbiota depletion increased food intake, and the microbiota-derived anorectic effect became significant during the consumption of high-fat but not standard food. This anorectic effect correlated with a specific postprandial microbial metabolic signature, and did not require postprandial endotoxemia or an NOD-, LRR-, and Pyrin domain-containing protein 3-inflammasome-mediated inflammatory response. Innovation and Conclusion: These findings show that the gut microbiota controls host appetite at the time scale of a meal under normal physiology. Interestingly, a microbiota-derived anorectic effect develops specifically with a high-fat meal, indicating that gut microbiota activity is involved in the satietogenic properties of foods. Antioxid. Redox Signal. 37, 349-369.

Excretion, Metabolism, and Tissue Distribution of Gelsemium elegans (Gardn. & Champ.) Benth in Pigs.

Gelsemium elegans (Gardn. & Champ.) Benth is a toxic flowering plant in the family Loganiaceae used to treat skin diseases, neuralgia and acute pain. The high toxicity of G. elegans restricts its development and clinical applications, but in veterinary applications, G. elegans has been fed to pigs as a feed additive without poisoning. However, until now, the in vivo processes of the multiple components of G. elegans have not been studied. This study investigates the excretion, metabolism and tissue distribution of the multiple components of G. elegans after feeding it to pigs in medicated feed. Pigs were fed 2% G. elegans powder in feed for 45 days. The plasma, urine, bile, feces and tissues (heart, liver, lung, spleen, brain, spinal cord, adrenal gland, testis, thigh muscle, abdominal muscle and back muscle) were collected 6 h after the last feeding and analyzed using high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Five natural products in plasma, twelve natural products and five metabolites in urine, and three natural products in feces were characterized, suggesting that multiple components from G. elegans were excreted in the urine. However, ten natural products and four metabolites were detected in bile samples, which suggested that G. elegans is involved in enterohepatic circulation in pigs. A total of seven of these metabolites were characterized, and four metabolites were glucuronidated metabolites. Ten natural products and six metabolites were detected in the tissues, which indicates that G. elegans is widely distributed in tissues and can cross the blood-brain barrier. Among the characterized compounds, a highly toxic gelsedine-type alkaloid from G. elegans was the main compound detected in all biological samples. This is the first study of the excretion, metabolism and tissue distribution of multiple components from G. elegans in pigs. These data can provide an important reference to explain the efficacy and toxicity of G. elegans. Additionally, the results of the tissue distribution of G. elegans are of great value for further residue depletion studies and safety evaluations of products of animals fed G. elegans.

Pharmacokinetics and Tissue Residue Profiles of Enrofloxacin in Crucian Carp (Carassius auratus gibelio) Following Single and Multiple Oral Administration.

The pharmacokinetics, tissue distribution, and elimination of enrofloxacin (ENR) and its metabolite ciprofloxacin (CIP) were investigated to the crucian carp (Carassius auratus gibelio) after single (20 mg/kg b. w.) and multiple oral administration (20 mg/kg b.w. one time daily for 5 days) at 28°C. The concentrations of ENR and CIP in the plasma and tested tissues (muscle/skin, liver, and kidney) were detected simultaneously by high-performance liquid chromatography (HPLC), and the pharmacokinetic data were analyzed with a non-compartmental model using WinNonLin 6.1 PK software (Pharsight Corporation, Mountain View, CA, USA). The pharmacokinetic characteristics of ENR in crucian carp exhibited slow absorption, wide tissue distribution, and long elimination half-life. In the single-dose group, the peak concentrations (Cmax) of ENR in the plasma, muscle/skin, liver, and kidney were 8.93 μg/mL, 13.9 μg/g, 31.2 μg/g, and 27.3 μg/g, respectively, observed at 3 h, 6 h, 1 h, and 3 h after dosing. The elimination half-lives (T1/2λz) of ENR in plasma, muscle/skin, liver, and kidney were calculated to be 67.4, 82.8, 94.4, and 114 h, respectively. In the multiple-dose group, the Cmax of ENR in the plasma, muscle/skin, liver, and kidney were 18.4 μg/mL, 26.8 μg/g, 82.8 μg/g, and 74.5 μg/g, respectively, achieved at 3 h, 6 h, 1 h, and 1 h after the last dose. The T1/2λz of ENR in the plasma, muscle/skin, liver, and kidney were calculated to be 76.4 h, 91.5 h, 114 h, and 148 h, respectively. During the multiple-dose administration, significant accumulations of ENR and CIP were observed in the plasma and tissues of crucian carp, possibly due to their long elimination half-lives. In both dose groups, the AUC0−∞ for both ENR and CIP followed the order of liver > kidney > muscle/skin > plasma. The finding suggested that the liver may play an important role in the metabolism of ENR. According to the calculated PK/PD indices of Cmax/minimum inhibitory concentrations (MIC) and AUC24h/MIC, the multiple-dose regimen would be highly effective against pathogenic bacteria with a MIC value of ≤ 1.84 μg/ml. Depletion studies indicated that a withdrawal period of at least 29 or 32 days was necessary to guarantee food security after single or multiple oral gavage administration at 28°C.

In Vivo Administration of Stanozolol in Cattle: Depletion and Stability Studies Using UHPLC-Q-Orbitrap.

An in vivo study was performed in order to evaluate the depletion time of stanozolol and its main metabolites using naturally incurred urine sample collected after the administration of intramuscular injections in 12 steers. A stability study was also carried out to investigate the influence of the storage period and the freeze-thaw cycles. A fast parent drug metabolization was observed, because within 6 h after drug administration, the signal of the metabolite 16β-hydroxystanozolol was predominant. After the second drug administration, a detection window of 17 days was obtained. The stability was studied using ANOVA, in which a storage condition of -20 °C proved stable during 240 days, which was also confirmed after 5 freeze-thaw cycles.

P52 signaling promotes cellular senescence

BackgroundNuclear factor-κB is a multi-subunit transcription factor that plays a central role in cellular senescence. We previously reported that an increase in the p52 subunit is seen in senescent cells and aged tissue. In the current work, we examined the mechanism by which p52 is activated and whether the increase in p52 promotes senescence.ResultsUsing both primary mouse embryonic fibroblasts (MEFs) and WI-38 human lung fibroblasts, we examined cells after serial passage and following prolonged culture. An increase in p52 was found in the nucleus relative to pre-senescent cells. The increase in p52 protein was not reflected by an increase in NFKB2 mRNA or by an increase in the abundance of upstream activating kinases, IKKα and NIK. To examine whether p52 promotes senescence, we over-expressed mature p52 in primary MEFs. Significantly more senescence was seen compared to control, a finding not seen with p52 mutated at critical DNA binding residues. In addition, blocking p52 nuclear translocation with the peptide inhibitor, SN52, decreased β-galactosidase (β-gal) formation. Subsequent filtration studies demonstrated that proteins in conditioned media (CM) were necessary for the increase in p52 and mass spectrometry identified S100A4 and cyclophilin A (CYPA) as potential factors in CM necessary for induction of p52. The requirement of these proteins in CM for induction of p52 was confirmed using depletion and supplementation studies. In addition, we found that activation of STAT3 signaling was required for the increase in p52. Finally, genome wide ChIP-sequencing analysis confirmed that there is an increase in p52 chromatin enrichment with senescence and identified several downstream factors whose expression is regulated by increased p52 binding.ConclusionsThese results demonstrate that p52 nuclear translocation is increased in senescent cells by factors in conditioned media and that mature p52 induces cellular senescence. The data are consistent with the prior observation that p52 is elevated in aged tissue and support the hypothesis that p52 contributes to organismal aging.

Development of disulfide-functionalized peptides covalently binding G protein-coupled receptors

A broadly applicable synthesis of peptides incorporating mixed disulfides between cysteine and homocysteine and cysteamine was developed. The method was established using pharmacologically relevant G protein-coupled receptor (GPCR) ligands including the μ-receptor agonist Dmt-DALDA and extended to the orexin derivative Oxa(17–33) and NT(8–13), the C-terminal hexapeptide of neurotensin. The newly developed NT(8–13) analog 6b incorporating an S-functionalized homocysteine revealed covalent binding of the neurotensin receptor 1 (NTSR1) in a radioligand depletion study.

Clonotypic analysis of protective influenza M2e-specific lung resident Th17 memory cells reveals extensive functional diversity

The fate of tissue-resident memory CD4 T cells (Trm) has been incompletely investigated. Here we show that intranasal, but not parenteral, immunization with CTA1-3M2e-DD stimulated M2e-specific Th17 Trm cells, which conferred strong protection against influenza virus infection in the lung. These cells rapidly expanded upon infection and effectively restricted virus replication as determined by CD4 T cell depletion studies. Single-cell RNAseq transcriptomic and TCR VDJ-analysis of M2e-tetramer-sorted CD4 T cells on day 3 and 8 post infection revealed complete Th17-lineage dominance (no Th1 or Tregs) with extensive functional diversity and expression of gene markers signifying mature resident Trm cells (Cd69, Nfkbid, Brd2, FosB). Unexpectedly, the same TCR clonotype hosted cells with different Th17 subcluster functions (IL-17, IL-22), regulatory and cytotoxic cells, suggesting a tissue and context-dependent differentiation of reactivated Th17 Trm cells. A gene set enrichment analysis demonstrated up-regulation of regulatory genes (Lag3, Tigit, Ctla4, Pdcd1) in M2e-specific Trm cells on day 8, indicating a tissue damage preventing function. Thus, contrary to current thinking, lung M2e-specific Th17 Trm cells are sufficient for controlling infection and for protecting against tissue injury. These findings will have strong implications for vaccine development against respiratory virus infections and influenza virus infections, in particular.

TUSC2 immunogene enhances efficacy of chemo-immuno combination on KRAS/LKB1 mutant NSCLC in humanized mouse model

KRAS/LKB1 (STK11) NSCLC metastatic tumors are intrinsically resistant to anti-PD-1 or PD-L1 immunotherapy. In this study, we use a humanized mouse model to show that while carboplatin plus pembrolizumab reduce tumor growth moderately and transiently, the addition of the tumor suppressor gene TUSC2, delivered systemically in nanovesicles, to this combination, eradicates tumors in the majority of animals. Immunoprofiling of the tumor microenvironment shows the addition of TUSC2 mediates: (a) significant infiltration of reconstituted human functional cytotoxic T cells, natural killer cells, and dendritic cells; (b) induction of antigen-specific T cell responses; (c) enrichment of functional central and memory effector T cells; and (d) decreased levels of PD-1+ T cells, myeloid-derived suppressor cells, Tregs, and M2 tumor associated macrophages. Depletion studies show the presence of functional central and memory effector T cells are required for the efficacy. TUSC2 sensitizes KRAS/LKB1 tumors to carboplatin plus pembrolizumab through modulation of the immune contexture towards a pro-immune tumor microenvironment.

Abstract P3-11-01: Oncolytic herpes simplex virus immunovirotherapy eradicates breast cancer and prevents tumor relapse

Abstract Syngeneic mouse tumor models are critical to investigating new immunotherapeutic modalities and providing a rationale for clinical translation. Oncolytic herpes simplex virus (oHSV) is an emerging treatment strategy for breast cancer. Genetically modified oHSV selectively replicates in cancer cells (sparring healthy cells), induces immunogenic cancer cell death, and stimulates innate/adaptive anti-tumor immune responses. The therapeutic success of oHSV depends on a dynamic interaction between oHSV, the tumor, and the host. Here, we evaluated the anti-tumor effects of G47Δ-IL12, a genetically engineered oHSV expressing interleukin-12 (IL-12), in two orthotopic immunocompetent mouse models of breast cancer, E0771 (some classified this model as luminal B subtype and some as triple-negative) and EMT6 (triple-negative subtype), which are syngeneic to C57BL/6 and BALB/c mice, respectively. Intratumoral G47Δ-IL12 treatment led to complete eradication of orthotopic E0771 and EMT6 mammary tumors and prevention of lung metastases, resulting in 100% long-term survivors, which remained tumor-free during the observation period (>100 days following the last G47Δ-IL12 treatment). In a contralateral orthotopic mammary tumor model, G47Δ-IL12 treatment of primary tumors led to the eradication of both treated and untreated distal tumors, confirming the abscopal and anti-metastatic effect of G47Δ-IL12 virotherapy. All cured mice from G47Δ-IL12-treated groups rejected lethal tumor re-challenge in the contralateral naïve mammary fat pad, indicating successful generation of a protective memory response. In both E0771 and EMT6 models, cell-specific IFN-γ responses and the presence of effector memory T cells (CD4+CD44highCD62Llow phenotype) in spleens of long-term survivors, possibly contributing to tumor relapse prevention. G47Δ-IL12-mediated anti-tumor efficacy was associated with significant increases in CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) (vs. mock group) in both E0771 and EMT6 tumor lesions. G47Δ-IL12 monotherapy also led to a significantly increased CD8+/FoxP3+ ratio (an important parameter for immunotherapy success) in both tumor models. Interestingly, immune cell depletion studies demonstrated that efficacy requires both CD4+ and CD8+ T cells in the E0771 model while only CD8+ T cells were required in the EMT6 model. Conclusions: G47Δ-IL12 monotherapy was effective in eradicating both orthotopic and metastatic tumors and generating long-term protective memory responses in two syngeneic breast cancer models. Treatment efficacy of G47Δ-IL12 was associated with significant immunologic alterations of the TME. G47Δ-IL12 utilized distinct immunologic mechanisms of action to eradicate tumors: CD8+ T cell-dependent action in the EMT6 model, whereas CD4+ and CD8+ T cell-dependent efficacy in the E0771 model. Altogether, our data suggest that G47Δ-IL12 is an efficient inflammatory modulator and beneficially exploits the immune system to eradicate breast tumors. These studies provide the necessary supporting evidence for the clinical translation of this agent into breast cancer patients. Citation Format: Hong-My Nguyen, Othon Almanza, Dipongkor Saha. Oncolytic herpes simplex virus immunovirotherapy eradicates breast cancer and prevents tumor relapse [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-11-01.

Pharmacokinetics and Tissue Distribution of Enrofloxacin Following Single Oral Administration in Yellow River Carp (Cyprinus carpio haematoperus).

The pharmacokinetics and tissue distribution of enrofloxacin were determined in Yellow River carp (Cyprinus carpio haematopterus) reared at 20°C after single oral administration of enrofloxacin at 10 mg·kg−1 body weight (BW). Plasma, bile, and different tissue samples, including liver, kidney, gill, gut, and skin-muscle, were collected at predetermined times points. An HPLC method was developed to simultaneously determine the concentrations of enrofloxacin and its metabolite, ciprofloxacin. However, ciprofloxacin was only detectable in some liver samples with trace levels. Then the average enrofloxacin concentrations vs. time data were subjected to a non-compartmental analysis using WinNonLin 5.2 software. Multiple peaking profiles were observed in all enrofloxacin concentration-time curves. The peak concentration (Cmax) values were observed as 0.79, 1.01, 2.09, 2.85, 4.34, 10.78, and 13.07 μg·ml−1 (or g−1) in plasma, skin-muscle, gill, kidney, liver, bile, and gut, respectively, and the corresponding time to reach peak concentration (Tmax) was 8, 8, 1, 8, 1, 72, and 4 h, respectively. The values of elimination half-life (T1/2λZ) of enrofloxacin in different tissues was in the following order: gill (291.13 h) > liver (222.29 h) > kidney (157.22 h) > plasma (129.44 h) > gut (91.47 h) > skin-muscle (87.77 h) > bile (86.22 h). The present results showed that enrofloxacin had a wide distribution in different tissues, however slow absorption and elimination in Yellow River carp. Additionally, enrofloxacin exhibited large distribution in bile, indicating that bile excretion might be the primary elimination route of enrofloxacin in Yellow River carp. A withdrawal period was calculated as 379.2 °C-day for single oral dosing of enrofloxacin at 10 mg/kg BW. Based on the calculated PK/PD indices of AUC/MIC or Cmax/MIC, the current enrofloxacin dosing regimen might have a positive therapeutic effect on the infection of Flavobacterium columnare, Aeromonas sobria, or Aeromonas hydrophila. However, the depletion study following multiple oral doses should be carried out in Yellow River carp reared at lower temperatures, and the withdrawal period should also be further calculated.

Determination and Identification of Nicarbazin, Measured as 4,4'-Dinitrocarbanilide (DNC), in Chicken Tissues by Liquid Chromatography With Tandem Mass Spectrometry: Final Action 2013.07.

BackgroundAOAC Method 2013.07 was adopted as First Action in 2013. Since then, the method has been used in numerous residue depletion studies with favorable comments from analysts.ObjectiveTo analyze data from residue depletion studies to support Final Action status.MethodTen residue depletion studies were conducted during May 2014 through May 2019. For each study, harvested incurred tissues were analyzed for nicarbazin using AOAC Method 2013.07 in 1 of 4 laboratories. Each analytical run included one or more fortified quality control test portions. The data from these known fortified matrix test portions were analyzed for reproducibility and repeatability.ResultsFor muscle tissues, relative recovery was 90.4% (95% CI 83.8 to 97.5); RSDr was 5.4% (95% CI 3.8 to 9.2); and RSDR was 7.9%. In the liver, values were 94.5% (95% CI 91.1 to 98.0), 5.8% (95% CI 4.1 to 9.9), and 6.8%, respectively. In the kidney, values were 91.5% (95% CI 85.3 to 98.1), 5.2% (95% CI 3.7 to 8.8), and 9.0%, respectively. In skin with adhering fat, values were 94.5% (95% CI 89.2 to 100.1), 8.9% (95% CI 6.3 to 15.1), and 8.9%, respectively. In all cases, repeatability and reproducibility were within acceptable limits.ConclusionsThe data and positive feedback support the transition of AOAC Method 2013.07 from First Action to Final Action.HighlightsFinal action status is supported by data collected during routine use of the method rather than a traditional multi-laboratory collaborative study. Data were subjected to statistical analysis using the pC-metamer, and then transformed back to the traditional C-metamer.

Determination of zilpaterol in a residue depletion study using LC-MS/MS in cattle plasma, muscle, liver and kidney

Zilpaterol is a β-agonist compound which promotes fat loss and muscle gain in cattle, providing economic benefits. However, zilpaterol residues in the animal might introduce a significant risk to humans after consumption. In the present manuscript, a highly specific, sensitive method using Selected Reaction Monitoring (SRM) in positive electrospray ionization (ESI +) mode by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS) for plasma, muscle, liver and kidney is presented. For method development, composition of the aqueous mobile phase, precipitation agent, and solid phase extraction (SPE) conditions were optimized. The method was fully validated showing a good linearity and recovery average greater than or equal to 97 % for all matrices. The method was applied to residue depletion studies in cattle after withdrawal of zilpaterol supplementation at 3, 4, 5 and 6 days showing that tissues can be consumed by humans after 4th day of zilpaterol withdrawal.

An overlooked subset of Cx3cr1wt/wt microglia in the Cx3cr1CreER-Eyfp/wt mouse has a repopulation advantage over Cx3cr1CreER-Eyfp/wt microglia following microglial depletion

BackgroundFluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia.MethodsImmunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1CreER-Eyfp/wt mouse brains. Genetically mediated microglia depletion using Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies.ResultsWe unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre−Eyfp− microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1highCre−Eyfp− microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1highCre−Eyfp− microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1highCre−Eyfp− microglia are Cx3cr1wt/wtCre−Eyfp−. Finally, we demonstrated that CX3CL1–CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro.ConclusionsOur results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.

Cellobiose inhibits the release of deoxynivalenol from transformed deoxynivalenol-3-glucoside from Lactiplantibacillus plantarum

The masked mycotoxin deoxynivalenol-3-glucoside (D3G) has been reported to be a detoxification product in plants, but can be hydrolyzed into its toxic precursor, deoxynivalenol (DON). Herein, we reported that Lactiplantibacillus plantarum (L. plantarum) NMM.1, isolated from Inner Mongolia raw milk, can efficiently transform D3G to DON in a short time. The global transcriptome microarray profiling of L. plantarum NMM.1 revealed differential expression of genes related to the phosphotransferase system (PTS) when D3G was used as the sole carbohydrate source. By adding an exogenous carbon source, we found that cellobiose efficiently inhibited the conversion of D3G into its precursor toxin by L. plantarum NMM.1. Overall, substrate depletion studies, transcriptome analysis, and carbohydrate intervention studies of L. plantarum NMM.1 suggested that cellobiose could be used to prevent the transformation of D3G into its free native DON by L. plantarum, thereby preventing harm to the human body.

A Study of groundwater depletion and its impact on agriculture in Eastern Haryana

A Study of groundwater depletion and its impact on agriculture in Eastern Haryana