A novel alcohol+nicotine co-use self-administration procedure reveals sex differences and differential alteration of mesocorticolimbic TLR- and cholinergic-related neuroimmune gene expression in rats.

Although alcohol and nicotine are two of the most commonly co-used drugs with upwards of 90% of adults with an alcohol use disorder (AUD) in the US also smoking, we don't tend to study alcohol and nicotine use this way. The current studies sought to develop and assess a novel alcohol + nicotine co-access self-administration (SA) model in adult male and female Long-Evans rats. Further, both drugs are implicated in neuroimmune function, albeit in largely opposing ways. Chronic alcohol use increases neuroinflammation via toll-like receptors (TLRs) which in turn increases alcohol intake. By contrast, nicotine produces anti-inflammatory effects, in part, through the monomeric alpha7 receptor (ChRNa7). Following long-term co-access (6 months), rats reliably administered both drugs during daily sessions, however males generally responded for more alcohol and females for nicotine. This was reflected in plasma analysis with translationally relevant intake levels of both alcohol and nicotine, making it invaluable in studying the effects of co-use on behavior and CNS function. Moreover, male rats show sensitivity to alterations in alcohol concentration whereas females show sensitivity to alterations in nicotine concentration. Rats trained on this procedure also developed an anxiogenic phenotype. Finally, we assessed alterations in neuroimmune-related gene expression in the medial prefrontal cortex - prelimbic, (mPFC-PL), nucleus accumbens core (AcbC), and ventral tegmental area (VTA). In the AcbC, where α7 expression was increased and ß2 was decreased, markers of pro-inflammatory activity were decreased, despite increases in TLR gene expression suggesting that co-use with nicotine modulates inflammatory state downstream from the receptor level. By contrast, in mPFC-PL where α7 was not increased, both TLRs and downstream proinflammatory markers were increased. Taken together, these findings support that there are brain regional and sex differences with co-use of alcohol + nicotine SA and suggest that targeting nicotinic α7 may represent a novel strategy for treating alcohol + nicotine co-dependence.

Deciphering the anammox microbial community succession with humic acid exposure to optimize large anammox granules for robust nitrogen removal.

The robustness of the anaerobic ammonia oxidation (anammox) process in treating wastewater with high concentrations of humic acids (HAs), including landfill leachate and sludge anaerobic digestion liquid, has been paid great attention. This study revealed that the anammox sludge granule size of 1.0-2.0 mm could be robust under the HA exposure with high concentrations. The total nitrogen removal efficiency (NRE) was 96.2% at the HA concentration of 20-100 mg/L, while the NRE was 88.5% at the HA concentration of 500 mg/L, with reduced by 7.7%. The increased extracellular polymeric substances (EPS) content which was stimulated by the HA exposure favored the formation of large granules (1.0-2.0 mm) by enveloping medium and micro granules (0.2-1.0 mm). The abundance of anammox bacteria Candidatus Brocadia was found to be higher (14.2%) in large anammox granules sized 1.0-2.0 mm, suggesting a potentially high anammox activity. However, the abundance of denitrifiers Denitratisoma increased by 4.3% in ultra-large anammox granules sized >2.0 mm, which could be attributed to the high EPS content for heterotrophic denitrifiers metabolism as organic matter. The feedback mechanism of the anammox community for maintaining the ecological function under the HA exposure resulted in a closely related microbial community, with positive and negative correlations in the ecological network increased by 64.3%. This study revealed that the HA exposure of the anammox system resulted in the anammox granules of 1.0-2.0 mm size being the dominant granules with robust nitrogen removal, providing significant guidance for the optimization of anammox granules for an efficient treatment of HA-containing wastewater in anammox applications.

Effects of cannabidiol, with and without ∆9-tetrahydrocannabinol, on anxiety-like behavior following alcohol withdrawal in mice.

Introduction: Alcohol use disorder (AUD) is commonly associated with anxiety disorders and enhanced stress-sensitivity; symptoms that can worsen during withdrawal to perpetuate continued alcohol use. Alcohol increases neuroimmune activity in the brain. Our recent evidence indicates that alcohol directly modulates neuroimmune function in the central amygdala (CeA), a key brain region regulating anxiety and alcohol intake, to alter neurotransmitter signaling. We hypothesized that cannabinoids, such as cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), which are thought to reduce neuroinflammation and anxiety, may have potential utility to alleviate alcohol withdrawal-induced stress-sensitivity and anxiety-like behaviors via modulation of CeA neuroimmune function. Methods: We tested the effects of CBD and CBD:THC (3:1 ratio) on anxiety-like behaviors and neuroimmune function in the CeA of mice undergoing acute (4-h) and short-term (24-h) withdrawal from chronic intermittent alcohol vapor exposure (CIE). We further examined the impact of CBD and CBD:THC on alcohol withdrawal behaviors in the presence of an additional stressor. Results: We found that CBD and 3:1 CBD:THC increased anxiety-like behaviors at 4-h withdrawal. At 24-h withdrawal, CBD alone reduced anxiety-like behaviors while CBD:THC had mixed effects, showing increased center time indicating reduced anxiety-like behaviors, but increased immobility time that may indicate increased anxiety-like behaviors. These mixed effects may be due to altered metabolism of CBD and THC during alcohol withdrawal. Immunohistochemical analysis showed decreased S100ß and Iba1 cell counts in the CeA at 4-h withdrawal, but not at 24-h withdrawal, with CBD and CBD:THC reversing alcohol withdrawal effects.. Discussion: These results suggest that the use of cannabinoids during alcohol withdrawal may lead to exacerbated anxiety depending on timing of use, which may be related to neuroimmune cell function in the CeA.

Activity of DNA polymerase κ across the genome in human fibroblasts.

DNA polymerase κ (Polκ) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by Polκ utilizing a Polκ-specific substrate, N2-(4-ethynylbenzyl)-2'-deoxyguanosine (EBndG). After shearing of the DNA into 200 to 500 bp lengths, the EBndG-containing DNA was covalently bound to biotin using the Cu(I)-catalyzed alkyne-azide cycloaddition reaction and isolated with streptavidin beads. Isolated DNA was then ligated to adaptors, followed by PCR amplification and next-generation sequencing to generate genome-wide repair maps. We have termed this method polymerase κ sequencing. Here, we present the human genome maps for Polκ activity in an undamaged cell line. We found that Polκ activity was enhanced in GC-rich regions, euchromatin regions, the promoter of genes, and in DNA that is replicated early in the S phase.

PC Gene Affects Milk Production Traits in Dairy Cattle.

In previous work, we found that PC was differentially expressed in cows at different lactation stages. Thus, we deemed that PC may be a candidate gene affecting milk production traits in dairy cattle. In this study, we found the polymorphisms of PC by resequencing and verified their genetic associations with milk production traits by using an animal model in a cattle population. In total, we detected six single-nucleotide polymorphisms (SNPs) in PC. The single marker association analysis showed that all SNPs were significantly associated with the five milk production traits (p < 0.05). Additionally, we predicted that allele G of 29:g.44965658 in the 5' regulatory region created binding sites for TF GATA1 and verified that this allele inhibited the transcriptional activity of PC by the dual-luciferase reporter assay. In conclusion, we proved that PC had a prominent genetic effect on milk production traits, and six SNPs with prominent genetic effects could be used as markers for genomic selection (GS) in dairy cattle, which is beneficial for accelerating the improvement in milk yield and quality in Chinese Holstein cows.

Polymorphisms of CYP7A1 and HADHB Genes and Their Effects on Milk Production Traits in Chinese Holstein Cows.

Our preliminary research proposed the cytochrome P450 family 7 subfamily A member 1 (CYP7A1) and hydroxyacyl-coenzyme A dehydrogenase trifunctional multienzyme complex beta subunit (HADHB) genes as candidates for association with milk-production traits in dairy cattle because of their differential expression across different lactation stages in the liver tissues of Chinese Holstein cows and their potential roles in lipid metabolism. Hence, we identified single-nucleotide polymorphisms (SNPs) of the CYP7A1 and HADHB genes and validated their genetic effects on milk-production traits in a Chinese Holstein population with the goal of providing valuable genetic markers for genomic selection (GS) in dairy cattle, This study identified five SNPs, 14:g.24676921A>G, 14:g.24676224G>A, 14:g.24675708G>T, 14:g.24665961C>T, and 14:g.24664026A>G, in the CYP7A1 gene and three SNPs, 11:g.73256269T>C, 11:g.73256227A>C, and 11:g.73242290C>T, in HADHB. The single-SNP association analysis revealed significant associations (p value ≤ 0.0461) between the eight SNPs of CYP7A1 and HADHB genes and 305-day milk, fat and protein yields. Additionally, using Haploview 4.2, we found that the five SNPs of CYP7A1 formed two haplotype blocks and that the two SNPs of HADHB formed one haplotype block; notably, all three haplotype blocks were also significantly associated with milk, fat and protein yields (p value ≤ 0.0315). Further prediction of transcription factor binding sites (TFBSs) based on Jaspar software (version 2023) showed that the 14:g.24676921A>G, 14:g.24675708G>T, 11:g.73256269T>C, and 11:g.73256227A>C SNPs could alter the 5' terminal TFBS of the CYP7A1 and HADHB genes. The 14:g.24665961C>T SNP caused changes in the structural stability of the mRNA for the CYP7A1 gene. These alterations have the potential to influence gene expression and, consequently, the phenotype associated with milk-production traits. In summary, we have confirmed the genetic effects of CYP7A1 and HADHB genes on milk-production traits in dairy cattle and identified potential functional mutations that we suggest could be used for GS of dairy cattle and in-depth mechanistic studies of animals.

Methoxsalen Inhibits the Acquisition of Nicotine Self-Administration: Attenuation by Cotinine Replacement in Male Rats.

INTRODUCTION: Cigarette smoking remains the leading preventable cause of disease and death. Nicotine is the primary reinforcing ingredient in cigarettes sustaining addiction. Cotinine is the major metabolite of nicotine that produces a myriad of neurobehavioral effects. Previous studies showed that cotinine-supported self-administration in rats and rats with a history of cotinine self-administration exhibited relapse-like drug-seeking behavior, suggesting that cotinine may also be reinforcing. To date, whether cotinine may contribute to nicotine reinforcement remains unknown. Nicotine metabolism is mainly catalyzed by hepatic CYP2B1/2 enzymes in rats and methoxsalen is a potent CYP2B1/2 inhibitor. AIMS AND METHODS: The study examined nicotine metabolism, self-administration, and locomotor activity. The hypothesis is that methoxsalen inhibits nicotine self-administration and cotinine replacement attenuates the inhibitory effects of methoxsalen in male rats. RESULTS: Methoxsalen decreased plasma cotinine levels following a subcutaneous nicotine injection. Repeated daily methoxsalen treatments reduced the acquisition of nicotine self-administration, leading to fewer nicotine infusions, lower nicotine intake, and lower plasma cotinine levels. However, methoxsalen did not alter the maintenance of nicotine self-administration despite a significant reduction of plasma cotinine levels. Cotinine replacement by mixing cotinine with nicotine for self-administration dose-dependently increased plasma cotinine levels and enhanced the acquisition of self-administration. Neither basal nor nicotine-induced locomotor activity was altered by methoxsalen. CONCLUSIONS: These results indicate that methoxsalen inhibition of cotinine formation impaired the acquisition of nicotine self-administration, and cotinine replacement attenuated the inhibitory effects of methoxsalen on the acquisition of self-administration, suggesting that cotinine may contribute to the initial development of nicotine reinforcement. IMPLICATIONS: Smoking cessation medications targeting nicotine's effects are only moderately effective, making it imperative to better understand the mechanisms of nicotine misuse. Methoxsalen inhibited nicotine metabolism to cotinine and impaired the acquisition of nicotine self-administration. Cotinine replacement restored plasma cotinine and attenuated the methoxsalen inhibition of nicotine self-administration in rats. These results suggest that (1) the inhibition of nicotine metabolism may be a viable strategy in reducing the development of nicotine reinforcement, (2) methoxsalen may be translationally valuable, and (3) cotinine may be a potential pharmacological target for therapeutic development given its important role in the initial development of nicotine reinforcement.

Green synthesis of iron nanoparticles using mulberry leaf extract: characterization, identification of active biomolecules, and catalytic activity.

The synthesis of iron-based nanoparticles (Fe NPs) using traditional preparation methods suffered from the disadvantages of high cost, environmental harm, and easy agglomeration. In this study, a novel eco-friendly method was proposed for the synthesis of iron nanomaterials (ML-Fe NPs): using antioxidant components extracted from mulberry leaf to reduce divalent iron (II). The preparation conditions of ML-Fe NPs were optimized by orthogonal tests. The prepared ML-Fe NPs exhibited an amorphous core-shell structure, displaying excellent dispersion and stability. During the synthesis process of ML-Fe NPs, the polyphenol molecules in mulberry leaf extract played a dominant role. A possible synthetic mechanism involving complexation, reduction, and encapsulation was proposed. Furthermore, the ML-Fe NPs were utilized to construct an ML-Fe NPs/peroxymonosulfate catalytic system for the degradation of Rhodamine B dye wastewater. The ML-Fe NPs demonstrated remarkable catalytic potential, achieving a 99% degradation efficiency for Rhodamine B within a span of 40 min.

In Vitro and In Vivo Assessment of the Infection Resistance and Biocompatibility of Small-Molecule-Modified Polyurethane Biomaterials.

Bacterial intracellular nucleotide second messenger signaling is involved in biofilm formation and regulates biofilm development. Interference with the bacterial nucleotide second messenger signaling provides a novel approach to control biofilm formation and limit microbial infection in medical devices. In this study, we tethered small-molecule derivatives of 4-arylazo-3,5-diamino-1H-pyrazole on polyurethane biomaterial surfaces and measured the biofilm resistance and initial biocompatibility of modified biomaterials in in vitro and in vivo settings. Results showed that small-molecule-modified surfaces significantly reduced the Staphylococcal epidermidis biofilm formation compared to unmodified surfaces and decreased the nucleotide levels of c-di-AMP in biofilm cells, suggesting that the tethered small molecules interfere with intracellular nucleotide signaling and inhibit biofilm formation. The hemocompatibility assay showed that the modified polyurethane films did not induce platelet activation or red blood cell hemolysis but significantly reduced plasma coagulation and platelet adhesion. The cytocompatibility assay with fibroblast cells showed that small-molecule-modified surfaces were noncytotoxic and cells appeared to be proliferating and growing on modified surfaces. In a 7-day subcutaneous infection rat model, the polymer samples were implanted in Wistar rats and inoculated with bacteria or PBS. Results show that modified polyurethane significantly reduced bacteria by ∼2.5 log units over unmodified films, and the modified polymers did not lead to additional irritation/toxicity to the animal tissues. Taken together, the results demonstrated that small molecules tethered on polymer surfaces remain active, and the modified polymers are biocompatible and resistant to microbial infection in vitro and in vivo.

Gender Difference in DNA Damage Induced by the Environmental Carcinogen Dibenzo[def,p]chrysene Individually and in Combination with Mouse Papillomavirus Infection in the Mouse Oral Cavity.

Tobacco smoking and human papillomavirus infection are established etiological agents in the development of head and neck squamous cell carcinoma (HNSCC). The incidence and mortality of HNSCC are higher in men than women. To provide biochemical basis for sex differences, we tested the hypothesis that carcinogen treatment using dibenzo[def,p]chrysene, which is an environmental pollutant and tobacco smoke constituent, in the absence or presence of the mouse papillomavirus infection results in significantly higher levels of DNA damage in the oral cavity in male than in female mice. However, the results of the present investigation do not support our hypothesis since we found that females were more susceptible to carcinogen-induced covalent DNA damage than males independent of the viral infection. Since DNA damage represents only a single-step in the carcinogenesis process, additional factors may contribute to sex differences in humans.

The Effects of Black Raspberry as a Whole Food-Based Approach on Biomarkers of Oxidative Stress in Buccal Cells and Urine of Smokers.

Cigarette smoke is a rich source of free radicals that can promote oxidative stress and carcinogenesis, including head and neck squamous cell carcinoma (HNSCC) development; importantly, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-iso-prostaglandin F2α (8-isoprostane) are biomarkers of oxidative stress. Several mechanisms, including the antioxidant properties of black raspberry (BRB), account for their chemopreventive effects. In the present clinical trial, we tested the hypothesis that BRB administration reduces biomarkers levels of oxidative stress in buccal cells and urine of smokers. One week after enrolling 21 smokers, baseline buccal cells and urine samples were collected before the administration of BRB lozenges for 8 weeks (5/day, 1 gm BRB/lozenge). Buccal cells and urine samples were collected at the middle and the end of BRB administration. The last samples were collected after the BRB cessation (washout period). We analyzed levels of 8-oxodG and 8-isoprostane (LC/MS-MS), urinary cotinine (ELISA), and creatinine (spectrophotometry). BRB significantly reduced the levels of 8-oxodG by 17.08% (P = 0.00079) in buccal cells and 12.44% (P = 0.034) in urine at the middle of BRB administration as compared with baseline; the corresponding values at the end of BRB administration were 16.46% (P = 0.026) in buccal cells and 25.72% (P = 0.202) in urine. BRB had no significant effect on the levels of urinary 8-isoprostane. BRB's capacity to inhibit 8-oxodG formation of smokers' buccal cells and urine is clearly evident and the reduction in 8-oxodG suggests that antioxidant abilities are central to BRB's HNSCC chemopreventive properties. PREVENTION RELEVANCE: Cigarette smoke contains highly active components namely free radicals that can promote oxidative stress and oral cancer. We found that black raspberry (BRB) inhibited the formation of oxidative stress markers in the oral cavity and urine of smokers suggesting the antioxidant abilities of BRB in preventing oral cancer.

2-Deoxyglucose and hydroxychloroquine HPLC-MS-MS analytical methods and pharmacokinetic interactions after oral co-administration in male rats.

Our previous work has shown a synergistic tumoricidal efficacy of combining the hexokinase (HK) inhibitor 2-deoxyglucose (2-DG) and the autophagy inhibitor chloroquine (CQ) through intraperitoneal injections on HK2-addicted prostate cancers in animal models. The pharmacokinetic (PK) behaviors of these oral drugs after simultaneous oral administration have not been reported. We developed high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) analytical methods for 2-DG and the clinically favored drug hydroxychloroquine (HCQ) for sera samples. Using a jugular vein-cannulated male rat model with serial blood collection before and after a single gavage dose of each drug alone or in combination, we examined their PK metrics for drug-drug interactions. The data demonstrated a rapid and complete separation of 2-DG from common monosaccharides by HPLC-MS-MS multi-reaction monitoring. Application of the HPLC-MS-MS 2-DG and HCQ methods to sera samples of nine rats showed a peak time (Tmax ) for 2-DG of 0.5 h after 2-DG alone or with HCQ and identical post-peak half-life of approximately 1 h. With a seemingly bi-modal time course for HCQ, the Tmax for HCQ alone (1.2 h) was faster than that for the combination (2 h; p = .017). After combination dosing, the peak concentration (Cmax ) and area under the curve (AUC0-4h ) of 2-DG were decreased by 53.8% (p = .0004) and 53.7% (p = .0001), whereas AUC0-8h for HCQ was decreased by 30.8% (p = .0279) from the respective single dosing. Without changing the mean residence time (MRT0-∞ ) of each drug, the combination affected the apparent volume of distribution (Vd ) and clearance (CL) of 2-DG, and CL for HCQ without affecting its Vd . We observed significant negative PK interactions, probably at the intestinal absorption level, between 2-DG and HCQ taken simultaneously by mouth. Future optimization efforts are warranted for their combination regimen for clinical translation.

Identification of genetic associations and functional SNPs of bovine KLF6 gene on milk production traits in Chinese holstein.

BACKGROUND: Our previous research identified the Kruppel like factor 6 (KLF6) gene as a prospective candidate for milk production traits in dairy cattle. The expression of KLF6 in the livers of Holstein cows during the peak of lactation was significantly higher than that during the dry and early lactation periods. Notably, it plays an essential role in activating peroxisome proliferator-activated receptor α (PPARα) signaling pathways. The primary aim of this study was to further substantiate whether the KLF6 gene has significant genetic effects on milk traits in dairy cattle. RESULTS: Through direct sequencing of PCR products with pooled DNA, we totally identified 12 single nucleotide polymorphisms (SNPs) within the KLF6 gene. The set of SNPs encompasses 7 located in 5' flanking region, 2 located in exon 2 and 3 located in 3' untranslated region (UTR). Of these, the g.44601035G > A is a missense mutation that resulting in the replacement of arginine (CGG) with glutamine (CAG), consequently leading to alterations in the secondary structure of the KLF6 protein, as predicted by SOPMA. The remaining 7 regulatory SNPs significantly impacted the transcriptional activity of KLF6 following mutation (P < 0.005), manifesting as changes in transcription factor binding sites. Additionally, 4 SNPs located in both the UTR and exons were predicted to influence the secondary structure of KLF6 mRNA using the RNAfold web server. Furthermore, we performed the genotype-phenotype association analysis using SAS 9.2 which found all the 12 SNPs were significantly correlated to milk yield, fat yield, fat percentage, protein yield and protein percentage within both the first and second lactations (P < 0.0001 ~ 0.0441). Also, with Haploview 4.2 software, we found the 12 SNPs linked closely and formed a haplotype block, which was strongly associated with five milk traits (P < 0.0001 ~ 0.0203). CONCLUSIONS: In summary, our study represented the KLF6 gene has significant impacts on milk yield and composition traits in dairy cattle. Among the identified SNPs, 7 were implicated in modulating milk traits by impacting transcriptional activity, 4 by altering mRNA secondary structure, and 1 by affecting the protein secondary structure of KLF6. These findings provided valuable molecular insights for genomic selection program of dairy cattle.

Methoxsalen inhibited the acquisition of nicotine self-administration: attenuation by cotinine replacement in rats.

Cigarette smoking remains the leading preventable cause of disease and death. Nicotine is the primary reinforcing ingredient in cigarettes sustaining addiction. Cotinine is the major metabolite of nicotine that produces a myriad of neurobehavioral effects. Cotinine supported self-administration and rats with a history of intravenous self-administration of cotinine exhibited relapse-like drug-seeking behavior, suggesting cotinine may also be reinforcing. To date, a potential contribution of cotinine to nicotine reinforcement remains unknown. Nicotine metabolism is mainly catalyzed by hepatic CYP2B1 enzyme in the rat and methoxsalen is a potent CYP2B1 inhibitor. The study tested the hypothesis that methoxsalen inbibits nicotine metabolism and self-administration, and that cotinine replacement attenuates the inhibitory effects of methoxsalen. Acute methoxsalen decreased plasma cotinine levels and increased nicotine levels following subcutaneous nicotine injection. Repeated methoxsalen reduced the acquisition of nicotine self-administration, leading to fewer nicotine infusions, disruption of lever differentiation, smaller total nicotine intake, and lower plasma cotinine levels. On the other hand, methoxsalen did not alter nicotine self-administration during the maintenance phase despite great reduction of plasma cotinine levels. Cotinine replacement by mixing cotinine with nicotine for self-administration dose-dependently increased plasma cotinine levels, counteracted effects of methoxsalen, and enhanced the acquisition of self-administration. Neither basal nor nicotine-induced locomotor activity was altered by methoxsalen. These results indicate that methoxsalen depressed cotinine formation from nicotine and the acquisition of nicotine self-administration, and that replacement of plasma cotinine attenuated the inhibitory effects of methoxsalen, suggesting that cotinine may contribute to the development of nicotine reinforcement.

Surfaces modified with small molecules that interfere with nucleotide signaling reduce Staphylococcus epidermidis biofilm and increase the efficacy of ciprofloxacin.

Staphylococcus epidermidis are common bacteria associated with biofilm related infections on implanted medical devices. Antibiotics are often used in combating such infections, but they may lose their efficacy in the presence of biofilms. Bacterial intracellular nucleotide second messenger signaling plays an important role in biofilm formation, and interference with the nucleotide signaling pathways provides a possible way to control biofilm formation and to increase biofilm susceptibility to antibiotic therapy. This study synthesized small molecule derivates of 4-arylazo-3,5-diamino-1 H-pyrazole (named as SP02 and SP03) and found these molecules inhibited S. epidermidis biofilm formation and induced biofilm dispersal. Analysis of bacterial nucleotide signaling molecules showed that both SP02 and SP03 significantly reduced cyclic dimeric adenosine monophosphate (c-di-AMP) levels in S. epidermidis at doses as low as 25 µM while having significant effects on multiple nucleotides signaling including cyclic dimeric guanosine monophosphate (c-di-GMP), c-di-AMP, and cyclic adenosine monophosphate (cAMP) at high doses (100 µM or greater). We then tethered these small molecules to polyurethane (PU) biomaterial surfaces and investigated biofilm formation on the modified surfaces. Results showed that the modified surfaces significantly inhibited biofilm formation during 24 h and 7-day incubations. The antibiotic ciprofloxacin was used to treat these biofilms and the efficacy of the antibiotic (2 µg/mL) was found to increase from 94.8% on unmodified PU surfaces to > 99.9% on both SP02 and SP03 modified surfaces (>3 log units). Results demonstrated the feasibility of tethering small molecules that interfere with nucleotide signaling onto polymeric biomaterial surfaces and in a way that interrupts biofilm formation and increases antibiotic efficacy for S. epidermidis infections.

Nucleotide Messenger Signaling of Staphylococci in Responding to Nitric Oxide - Releasing Biomaterials.

Nitric oxide (NO) releasing biomaterials are a promising approach against medical device associated microbial infection. In contrast to the bacteria-killing effects of NO at high concentrations, NO at low concentrations serves as an important signaling molecule to inhibit biofilm formation or disperse mature biofilms by regulating the intracellular nucleotide second messenger signaling network such as cyclic dimeric guanosine monophosphate (c-di-GMP) for many Gram-negative bacterial strains. However, Gram-positive staphylococcal bacteria are the most commonly diagnosed microbial infections on indwelling devices, but much less is known about the nucleotide messengers and their response to NO as well as the mechanism by which NO inhibits biofilm formation. This study investigated the cyclic nucleotide second messengers c-di-GMP, cyclic dimeric adenosine monophosphate (c-di-AMP), and cyclic adenosine monophosphate (cAMP) in both Staphylococcus aureus (S. aureus) Newman D2C and Staphylococcus epidermidis (S. epidermidis) RP62A after incubating with S-nitroso-N-acetylpenicillamine (SNAP, NO donor) impregnated polyurethane (PU) films. Results demonstrated that NO release from the polymer films significantly reduced the c-di-GMP levels in S. aureus planktonic and sessile cells, and these bacteria showed inhibited biofilm formation. However, the effect of NO release on c-di-GMP in S. epidermidis was weak, but rather, S. epidermidis showed significant reduction in c-di-AMP levels in response to NO release and also showed reduced biofilm formation. Results strongly suggest that NO regulates the nucleotide second messenger signaling network in different ways for these two bacteria, but for both bacteria, these changes in signaling affect the formations of biofilms. These findings provide cues to understand the mechanism of Staphylococcus biofilm inhibition by NO and suggest novel targets for antibiofilm interventions.

2-Deoxyglucose and hydroxychloroquine HPLC-MS-MS analytical methods and pharmacokinetic interactions after oral co-administration in male rats.

Our previous work has shown a synergistic tumoricidal action of the hexokinase (HK) inhibitor 2-deoxyglucose (2-DG) and the autophagy inhibitor chloroquine (CQ) on HK2-addicted prostate cancers in animal models through intraperitoneal injections. Here we developed high performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) methods for 2-DG and clinically favored drug hydroxychloroquine (HCQ) and explored PK interaction of the orally administered drugs in a jugular vein cannulated male rat model, which allowed serial blood collection before and 0.5, 1, 2, 4 and 8 h after a single gavage dose of each drug alone or simultaneously after appropriate washout periods between the drugs. The results demonstrated a rapid and satisfactory separation of 2-DG standard from common monosaccharides by HPLC-MS-MS multi-reaction monitoring (MRM) and the presence of endogenous "2-DG". Application of the HPLC-MS-MS 2-DG and HCQ methods to sera samples of 9 evaluable rats showed a peak time (Tmax) of 2-DG of 0.5 h after 2-DG dosing alone or with HCQ and glucose-like PK behavior. With a seemingly bi-modal time course for HCQ, the Tmax for HCQ dosing alone (1.2 h) was faster than that for the combination (2 h; p = 0.013, 2-tailed t-test). After combination dosing, the peak concentration (Cmax) and area under the curve (AUC) of 2-DG were decreased by 54% (p < 0.0001) and 52%, whereas those for HCQ were decreased by 40% (p = 0.026) and 35%, respectively, compared to single dosing. The data suggest significant negative PK interactions between the two oral drugs taken simultaneously and warrant optimization efforts for the combination regimen.

Cannabinoids as Potential Cancer Therapeutics: The Concentration Conundrum.

Background: Studies have reported that cannabinoids, in particular Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), significantly reduce cancer cell viability in vitro. Unfortunately, treatment conditions vary significantly across reports. In particular, a majority of reports utilize conditions with reduced serum concentrations (0-3%) that may compromise the growth of the cells themselves, as well as the observed results. Objectives: This study was designed to test the hypothesis that, based on their known protein binding characteristics, cannabinoids would be less effective in the presence of fetal bovine serum (FBS). Moreover, we wished to determine if the treatments served to be cytotoxic or cytostatic under these conditions. Methods: Six cancer cell lines, representing two independent lines of three different types of cancer (glioblastoma, melanoma, and colorectal cancer [CRC]), were treated with 10 µM pure Δ9-THC, CBD, KM-233, and HU-331 for 48 h (in the presence or absence of FBS). Cell viability was measured with the MTT assay. Dose-response curves were then generated comparing the potencies of the four cannabinoids under the same conditions. Results: We found that serum-free medium alone produces cell cycle arrest for CRC cells and slows cell growth for the other cancer types. The antineoplastic effects of three of the four cannabinoids (Δ9-THC, CBD, and KM-233) increase when serum is omitted from the media. In addition, dose-response curves for these drugs demonstrated lower IC50 values for serum-free media compared with the media with 10% serum in all cell lines. The fourth compound, HU-331, was equally effective under both conditions. A further confound we observed is that omission of serum produces dramatic binding of Δ9-THC and CBD to plastic. Conclusions: Treatment of cancer cells in the absence of FBS appears to enhance the potency of cannabinoids. However, omission of FBS itself compromises cell growth and represents a less physiological condition. Given the knowledge that cannabinoids are 90-95% protein bound and have well-known affinities for plastic, it may be ill-advised to treat cells under conditions where the cells are not growing optimally and where known concentrations cannot be assumed (i.e., FBS-free conditions).

Diurnal variability of cortisol in the Pennsylvania adult smoking study: Exploration of association with nicotine intake.

Cortisol in saliva, urine and plasma follows a diurnal rhythm typically characterized as a morning peak and a decline throughout the waking day. While often measured under controlled conditions, inter-individual differences in cortisol diurnal rhythms in free living populations are not well characterized. Cortisol levels may vary substantially between individuals and the level of variation may differ depending on the time of day. Further, associations with individual characteristics such as nicotine dependence on cortisol rhythms have not been adequately determined. We developed a Liquid Chromatography-Tandem Mass Spectrometry method to measure cortisol in saliva of 180 smokers from the Pennsylvania Adult Smoking Study. Diurnal patters of cortisol were determined by obtaining five timed samples throughout the day for a total of 900 determinations. Adherence to the protocol was estimated by asking participants to record the time of sample collection. Longitudinal linear mixed effects models were developed to measure the predictors of mean levels. Phenotypic groups were constructed based on the minimum and maximum cortisol levels. Mixed method modelling was conducted to determine the effects of phenotype and study adherence as well as reported measures of stress, nicotine dependence and cigarette smoking frequency. Nicotine metabolites were measured to accurately quantify dose of smoking intake. Results showed that there was moderate compliance to the timed protocol. Descriptive and analytic findings showed that some smokers had atypical cortisol patterns, and that the cortisol profiles based on experience of maximum and minimum cortisol levels can predict how cortisol varies throughout the day. There was no association with reported stress, cigarettes per day, and nicotine metabolites. There was no association with the Fagerstrom Test for Nicotine Dependence. The Hooked on Nicotine Checklist (HONC) score was associated with decreased cortisol levels. Overall these results show new approaches and expectations to population-based studies of cortisol.

Longitudinal genome-wide association studies of milk production traits in Holstein cattle using whole-genome sequence data imputed from medium-density chip data.

Longitudinal traits, such as milk production traits in dairy cattle, are featured by having phenotypic values at multiple time points, which change dynamically over time. In this study, we first imputed SNP chip (50-100K) data to whole-genome sequence (WGS) data in a Chinese Holstein population consisting of 6,470 cows. The imputation accuracies were 0.88 to 0.97 on average after quality control. We then performed longitudinal GWAS in this population based on a random regression test-day model using the imputed WGS data. The longitudinal GWAS revealed 16, 39, and 75 quantitative trait locus regions associated with milk yield, fat percentage, and protein percentage, respectively. We estimated the 95% confidence intervals (CI) for these quantitative trait locus regions using the logP drop method and identified 581 genes involved in these CI. Further, we focused on the CI that covered or overlapped with only 1 gene or the CI that contained an extremely significant top SNP. Twenty-eight candidate genes were identified in these CI. Most of them have been reported in the literature to be associated with milk production traits, such as DGAT1, HSF1, MGST1, GHR, ABCG2, ADCK5, and CSN1S1. Among the unreported novel genes, some also showed good potential as candidate genes, such as CCSER1, CUX2, SNTB1, RGS7, OSR2, and STK3, and are worth being further investigated. Our study provided not only new insights into the candidate genes for milk production traits, but also a general framework for longitudinal GWAS based on random regression test-day model using WGS data.