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Coproporphyrin I (CPI) is an endogenous biomarker of OATP1B activity and associated drug‐drug interactions. In this study, a minimal physiologically‐based pharmacokinetic model was developed to investigate the impact of OATP1B1 genotype (c.521T>C), ethnicity, and sex on CPI pharmacokinetics and interindividual variability in its baseline. The model implemented mechanistic descriptions of CPI hepatic transport between liver blood and liver tissue and renal excretion. Key model parameters (e.g., endogenous CPI synthesis rate, and CPI hepatic uptake clearance) were estimated by fitting the model simultaneously to three independent CPI clinical datasets (plasma and urine data) obtained from white (n = 16, men and women) and Asian‐Indian (n = 26, all men) subjects, with c.521 variants (TT, TC, and CC). The optimized CPI model successfully described the observed data using c.521T>C genotype, ethnicity, and sex as covariates. CPI hepatic active was 79% lower in 521CC relative to the wild type and 42% lower in Asian‐Indians relative to white subjects, whereas CPI synthesis was 23% higher in male relative to female subjects. Parameter sensitivity analysis showed marginal impact of the assumption of CPI synthesis site (blood or liver), resulting in comparable recovery of plasma and urine CPI data. Lower magnitude of CPI‐drug interaction was simulated in 521CC subjects, suggesting the risk of underestimation of CPI‐drug interaction without prior OATP1B1 genotyping. The CPI model incorporates key covariates contributing to interindividual variability in its baseline and highlights the utility of the CPI modeling to facilitate the design of prospective clinical studies to maximize the sensitivity of this biomarker. [ABSTRACT FROM AUTHOR]

Staphylococcus pseudintermedius can cause severe infections of the skin, ear and other tissues in dogs and cats. Methicillin-resistant S. pseudintermedius (MRSP) has recently become more prevalent, posing a severe threat to companion animals and public health. Therefore, rapid and accurate diagnosis of S. pseudintermedius and MRSP infections in dogs and cats is essential for timely controlling infections. The development of CRISPR/Cas technology offers an innovative solution for rapid diagnosis. Here, we established an assay combining recombinant polymerase amplification (RPA) and CRISPR/Cas12a. By separately detecting the spsJ gene, the specific gene of S. pseudintermedius, and the mecA gene, the methicillin resistance gene, this method allows for the direct detection of methicillin-susceptible S. pseudintermedius (MSSP) and MRSP in clinical samples at 37 °C for a total of 40 min, The results can be directly visualized by the naked eye under blue light. The limits of detection of the RPA-CRISPR/Cas12a assay were 103 copies per reaction for the spsJ gene and 104 copies per reaction for the mecA gene. The RPA-CRISPR/Cas12a detection successfully detected and differentiated clinical isolates of MSSP and MRSP without cross-reactivity with other tested bacteria species. The evaluation of the detection performance of RPA-CRISPR/Cas12a with 47 clinical samples (without culture) from dogs and cats showed that the results of detection were 100% consistent with those of clinical culture and colony sequencing, which was more sensitive than PCR. RPA-CRISPR/Cas12a assay can quickly and sensitively detect S. pseudintermedius and MRSP in clinical samples without expensive instruments, making it suitable for small veterinary clinics. [ABSTRACT FROM AUTHOR]

BMS-932481 was designed to modulate ɣ-secretase activity to produce shorter and less amyloidogenic peptides, potentially averting liabilities associated with complete enzymatic inhibition. Although it demonstrated the intended pharmacology in the clinic, BMS-932481 unexpectedly caused drug-induced liver injury (DILI) in a multiple ascending dose study characterized by dose- and exposure-dependence, delayed onset manifestation, and a high incidence of hepatocellular damage. Retrospective studies investigating the disposition and probable mechanisms of toxicity of BMS-932481 are presented here. These included a mass balance study in bile-duct-cannulated rats and a metabolite profiling study in human hepatocytes, which together demonstrated oxidative metabolism followed by biliary elimination as the primary means of disposition. Additionally, minimal protein covalent binding in hepatocytes and lack of bioactivation products excluded reactive metabolite formation as a probable toxicological mechanism. However, BMS-932481 and 3 major oxidative metabolites were found to inhibit the bile salt export pump (BSEP) and multidrug resistance protein 4 (MRP4) in vitro. Considering human plasma concentrations, the IC50 values against these efflux transporters were clinically meaningful, particularly in the high dose cohort. Active uptake into human hepatocytes in vitro suggested the potential for hepatic levels of BMS-932481 to be elevated further above plasma concentrations, enhancing DILI risk. Conversely, measures of mitochondrial functional decline in hepatocytes treated with BMS-932481 were minimal or modest, suggesting limited contributions to DILI. Collectively, these findings suggested that repeat administration of BMS-932481 likely resulted in high hepatic concentrations of BMS-932481 and its metabolites, which disrupted bile acid transport via BSEP and MRP4, elevating serum biomarkers of liver injury. [ABSTRACT FROM AUTHOR]

In this work, a simple method is proposed to simultaneously reduce the size and cross-polarization level (XPL) of quarter-wave microstrip antennas by reconstructing the magnetic currents along the radiation edges. A design example of five shorted elements is given with a comparison of the quarter-wave microstrip antennas at 1.88 GHz. Measured results show an over 64% size reduction and 17-dB cross-polarization suppression enhancement for the proposed scheme as compared with the conventional one. Moreover, the proposed scheme exhibits good broadside radiation patterns with a gain of 5.4 dBi and the half-power beamwidths of about 104° in the E-plane and 84° in the H-plane. [ABSTRACT FROM AUTHOR]

A novel fan-out wafer-level packaging (FOWLP) technology with double-sided redistribution layers (RDLs) and tall copper vertical interconnects is described. The design of a dual-polarized magnetoelectric (ME) dipole antenna based on this new packaging technology is presented. It is shown that the antenna with a size of $10\times10$ mm2 achieves good impedance matching and a stable radiation pattern over a wide bandwidth from 25 to 43 GHz, which can cover most of the millimeter-wave (mmWave) bands of the fifth-generation (5G) mobile networks in different countries. The measurement results demonstrate that the antenna is well suited for FOWLP technology and can be used for phased-array modules in 5G wireless communication systems. [ABSTRACT FROM AUTHOR]

Mutations in S and 3c genes of feline coronavirus (FCoV) have been associated with the development of feline infectious peritonitis (FIP). In the present study, FCoV S and 3c genes mutations were analyzed in healthy and FIP cats. M1058L mutation was found in 13.64% (3/22) feces from FIP cats, but not in feces from healthy cats (0/39). The intact 3c gene was found in feces from both healthy cats (19/19) and FIP cats (12/12). All parenteral samples from FIP cats carried one or more of the M1058L mutation, S1060A mutation and mutated 3c gene. FCoV reverse-transcriptase polymerase chain reaction (RT-PCR) of parenteral samples (including ascites, pleural effusions and tissue) is recommended as the gold standard for clinical diagnosis of FIP rather than detection of the M1058L mutation, but when cats have severe gastrointestinal symptoms and lesions, detection of the M1058L mutation in feces may be helpful in diagnosing FIP. • FCoV in parenteral samples from FIP cats carried one or more of the M1058L mutation, S1060A mutation and mutations that lead to truncated 3c gene product. • FCoV reverse-transcriptase polymerase chain reaction (RT-PCR) of parenteral samples (including ascites, pleural effusions and tissue samples) is recommended as the gold standard for the clinical diagnosis of FIP rather than detection of the M1085L mutation. • When cats have severe gastrointestinal symptoms and lesions, detection of the M1058L mutation in feces may be helpful in diagnosing FIP, especially for some dry FIP without typical clinical symptoms of FIP. [ABSTRACT FROM AUTHOR]

Post-translational modifications of histone proteins greatly impact gene expression and cell fate decisions in eukaryotes. To study these, it is important to develop a convenient, multiplex, and efficient method to precisely introduce mutations to histones. Because eukaryotic cells usually contain multiple copies of histone genes, it is a challenge to mutate all histones at the same time by the traditional homologous recombination method. Here, we developed a CRISPR-Cas9 based shuffle system in Saccharomyces cerevisiae, to generate point mutations on both endogenous histone H3 and H4 genes in a rapid, seamless and multiplex fashion. Using this method, we generated yeast strains containing histone triple H3–K4R–K36R–K79R mutants and histone combinatorial H3–K56Q–H4–K59A double mutants with high efficiencies (70–80%). This CRISPR-Cas9 based mutagenesis system could be an invaluable tool to the epigenetics field. [ABSTRACT FROM AUTHOR]

Recent studies have focused on coproporphyrin (CP)-I and CP-III (CPs) as endogenous biomarkers for organic anion transporting polypeptides (OATPs). Previous data showed that CPs are also substrates of multidrug resistance-associated protein (MRP/Mrp) 2 and 3. This study was designed to examine the impact of loss of Mrp2 function on the routes of excretion of endogenous CPs in wild-type (WT) Wistar compared to Mrp2-deficient TR− rats. To exclude possible confounding effects of rat Oatps, the transport of CPs was investigated in Oatp-overexpressing HeLa cells. Results indicated that CPs are substrates of rodent Oatp1b2, and that CP-III is a substrate of Oatp2b1. Quantitative targeted absolute proteomic (QTAP) analysis revealed no differences in Oatps, but an expected significant increase in Mrp3 protein levels in TR− compared to WT rat livers. CP-I and CP-III concentrations measured by LC-MS/MS were elevated in TR− compared to WT rat liver, while CP-I and CP-III estimated biliary clearance was decreased 75- and 840-fold in TR− compared to WT rats, respectively. CP-III concentrations were decreased 14-fold in the feces of TR− compared to WT rats, but differences in CP-I were not significant. In summary, the disposition of CPs was markedly altered by loss of Mrp2 and increased Mrp3 function as measured in TR− rats. [ABSTRACT FROM AUTHOR]

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP 7) levels. 5-InsP 7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism. [Display omitted] • Hybrid of glycolysis with the phosphoketolase pathway made an efficient cell factory • Oca5, identified as an inositol pyrophosphatase, degrading 5-InsP 7 to InsP 6 • InsPs control gene expression involved in glycolysis and respiration to balance fluxes • The mechanism of Oca5 regulating glycolysis and respiration was elaborated Glycolysis is considered as the only energy-generating glucose catabolic pathway in eukaryotes. Qin et al. establish a hybrid-glycolysis yeast that disrupts the Embden-Meyerhof-Parnas glycolysis pathway and introduced components of the phosphoketolase pathway. They further identify Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration activities. [ABSTRACT FROM AUTHOR]

• The coexistence of Lewis acid/base sites in the IL-ZIF was confirmed. • The density of Lewis acid/base sites was tuned easily. • A high yield of 97% was achieved over an IL-ZIF catalyst. • Lewis acid/base sites reduce the energy barrier to 11.5 kcal mol−1. • A synergistic catalysis mechanism was confirmed by theoretical calculations. Ionic liquid (IL) immobilized zeolitic imidazolate framework (ZIF) catalysts (IL-ZIF-8) were prepared through grafting IL on a dual-ligand ZIF by a facile post-synthetic modification. The coordinately unsaturated Zn as Lewis acid sites and bromide ions as Lewis base sites were confirmed in the resulted IL-ZIF. The density of Lewis acid/base sites was tuned easily by content of mixed linkers in the framework and/or the IL loadings. The coexistence of Lewis acid/base sites remarkably improved the activity for cycloaddition of CO 2 with propylene oxide, compared with parent ZIFs and IL. In the absence of any co-catalyst and solvent, the prepared IL-ZIF-8(0.3) obtained a high yield of 97% with good stability and reusability. Besides, a Lewis acid/base synergistic catalytic mechanism was proposed. The synergetic interaction of Lewis acid sites and Lewis base sites significantly reduces energy barrier of propylene oxide ring-opening to 11.5 kcal mol−1 and thus promotes the CO 2 cycloaddition reaction. [ABSTRACT FROM AUTHOR]

The development of single-cell multi-omics technology has greatly enhanced our understanding of biology, and in parallel, numerous algorithms have been proposed to predict the protein abundance and/or chromatin accessibility of cells from single-cell transcriptomic information and to integrate various types of single-cell multi-omics data. However, few studies have systematically compared and evaluated the performance of these algorithms. Here, we present a benchmark study of 14 protein abundance/chromatin accessibility prediction algorithms and 18 single-cell multi-omics integration algorithms using 47 single-cell multi-omics datasets. Our benchmark study showed overall totalVI and scArches outperformed the other algorithms for predicting protein abundance, and LS_Lab was the top-performing algorithm for the prediction of chromatin accessibility in most cases. Seurat, MOJITOO and scAI emerge as leading algorithms for vertical integration, whereas totalVI and UINMF excel beyond their counterparts in both horizontal and mosaic integration scenarios. Additionally, we provide a pipeline to assist researchers in selecting the optimal multi-omics prediction and integration algorithm., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

We recently showed that riboflavin is a selected substrate of breast cancer resistance protein (BCRP) over P-glycoprotein (P-gp) and demonstrated its prediction performance in preclinical drug-drug interaction (DDI) studies. The aim of this study was to investigate the suitability of riboflavin to assess BCRP inhibition in humans. First, we assessed the substrate potential of riboflavin toward other major drug transporters using established transfected cell systems. Riboflavin is a substrate for organic anion transporter (OAT)1, OAT3, and multidrug and toxin extrusion protein (MATE)2-K, with uptake ratios ranging from 2.69 to 11.6, but riboflavin is not a substrate of organic anion-transporting polypeptide (OATP)1B1, OATP1B3, organic cation transporter (OCT)2, and MATE1. The effects of BMS-986371, a potent in vitro inhibitor of BCRP ( IC 50 0.40 μ M), on the pharmacokinetics of riboflavin, isobutyryl carnitine, and arginine were then examined in healthy male adults ( N = 14 or 16) after oral administration of methotrexate (MTX) (7.5 mg) and enteric-coated (EC) sulfasalazine (SSZ) (1000 mg) alone or in combination with BMS-986371 (150 mg). Oral administration of BMS-986371 increased the area under the plasma concentration-time curves ( AUC s) of rosuvastatin and immediate-release (IR) SSZ to 1.38- and 1.51-fold, respectively, and significantly increased AUC (0-4h), AUC (0-24h), and C max of riboflavin by 1.25-, 1.14-, and 1.11-fold ( P -values of 0.003, 0.009, and 0.025, respectively) compared with the MTX/SSZ EC alone group. In contrast, BMS-986371 did not significantly influence the AUC (0-24h) and C max values of isobutyryl carnitine and arginine (0.96- to 1.07-fold, respectively; P > 0.05). Overall, these data indicate that plasma riboflavin is a promising biomarker of BCRP that may offer a possibility to assess drug candidate as a BCRP modulator in early drug development. SIGNIFICANCE STATEMENT: Endogenous compounds that serve as biomarkers for clinical inhibition of breast cancer resistance protein (BCRP) are not currently available. This study provides the initial evidence that riboflavin is a promising BCRP biomarker in humans. For the first time, the value of leveraging the substrate of BCRP with acceptable prediction performance in clinical studies is shown. Additional clinical investigations with known BCRP inhibitors are needed to fully validate and showcase the utility of this biomarker., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Circular RNAs (circRNAs) exhibit unique patterns of expression and high levels of stability in patient plasma samples such that they represent ideal non-invasive biomarkers that can be leveraged to detect a wide array of diseases including endometrial cancer (EC). This study was designed to identify circRNAs with potential diagnostic utility in serum samples from EC patients while also evaluating the utility of macrophage migration inhibitory factor (MIF) as a biomarker when screening for this form of cancer in the clinic., Methods: Levels of circEPSTI1 and MIF were assessed in the plasma of EC patients and healthy subjects (n=186 each) through qPCR and ELISAs. The diagnostic utility of these biomarkers was assessed with receiver operating characteristic curve (ROC) analyses., Results: Relative to healthy subjects, EC patient serum contained significantly elevated circEPSTI1 and MIF. An association was noted between circEPSTI1 expression in stages, histologic grade, and residual tumor. ROC curves confirmed that serum circEPSTI1 levels distinguished between controls and patients with EC with an Area of 0.835 and serum MIF levels distinguished between controls and patients with EC with an Area of 0.6646. When instead diagnosing patients based on the combination of MIF and circEPSTI1, the Area further rose to 0.8604., Conclusion: Assessing the combination of circEPSTI1 and MIF may be a viable approach to reliably diagnosing EC., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Cui et al.)

Advancement of endogenous biomarkers for drug transporters as a tool for assessing drug-drug interactions (DDIs) depends on initial identification of biomarker candidates and relies heavily on biomarker validation and its response to reference inhibitors in vivo. To identify endogenous biomarkers of breast cancer resistance protein (BCRP), we applied metabolomic approaches to profile plasma from Bcrp -/- , multidrug resistance protein (Mdr)1a/1b -/- , and Bcrp/Mdr1a/1b -/- mice. Approximately 130 metabolites were significantly altered in Bcrp and P-glycoprotein (P-gp) knockout mice, indicating numerous metabolite-transporter interactions. We focused on BCRP-specific substrates and identified riboflavin, which was significantly elevated in the plasma of Bcrp single- and Bcrp/P-gp double- but not P-gp single-knockout mice. Dual BCRP/P-gp inhibitor elacridar caused a dose-dependent increase of the area under the plasma concentration-time curve ( AUC ) of riboflavin in mice (1.51- and 1.93-fold increases by 30 and 150 mg/kg elacridar, respectively). In three cynomolgus monkeys, we observed approximately 1.7-fold increases in the riboflavin concentrations caused by ML753286 (10 mg/kg), which correlated well with the increase of sulfasalazine, a known BCRP probe in monkeys. However, the BCRP inhibitor had no effect on isobutyryl carnitine, arginine, or 2-arachidonoyl glycerol levels. Additionally, clinical studies on healthy volunteers indicated low intrasubject and intermeal variability of plasma riboflavin concentrations. In vitro experiments using membrane vesicles demonstrated riboflavin as a select substrate of monkey and human BCRP over P-gp. Collectively, this proof-of-principle study indicates that riboflavin is a suitable endogenous probe for BCRP activity in mice and monkeys and that future investigation of riboflavin as a blood-based biomarker of human BCRP is warranted. SIGNIFICANCE STATEMENT: Our results identified riboflavin as an endogenous biomarker candidate of BCRP. Its selectivity, sensitivity, and predictivity regarding BCRP inhibition have been explored. The findings of this study highlight riboflavin as an informative BCRP plasma biomarker in animal models. The utility of this biomarker requires further validation by evaluating the effects of BCRP inhibitors of different potencies on riboflavin plasma concentrations in humans. Ultimately, riboflavin may shed light on the risk assessment of BCRP DDIs in early clinical trials., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

During yeast dough fermentation, such as the high-sucrose bread-making process, the yeast cells are subjected to considerable osmotic stress, resulting in poor outcomes. Invertase is important for catalyzing the irreversible hydrolysis of sucrose to free glucose and fructose, and decreasing the catalytic activity of the invertase may reduce the glucose osmotic stress on the yeast. In this study, we performed structural design and site-directed mutagenesis (SDM) on the Saccharomyces cerevisiae invertase (ScInV) in an Escherichia coli expression system to study the catalytic activity of ScInV mutants in vitro. In addition, we generated the same mutation sites in the yeast endogenous genome and tested their invertase activity in yeast and dough fermentation ability. Our results indicated that appropriately reduced invertase activity of yeast ScInV can enhance dough fermentation activity under high-sucrose conditions by 52%. Our systems have greatly accelerated the engineering of yeast endogenous enzymes both in vitro and in yeast, and shed light on future metabolic engineering of yeast., (© 2022. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

The aim of this study was to apply a strategy to express a recombinant CLP peptide and explore its application as a product derived from natural compounds. The amphiphilic CLP peptide was hybridized from three parent peptides (CM4, LL37, and TP5) and was considered to have potent endotoxin-neutralizing activity with minimal cytotoxic and hemolytic activity. To achieve high secretion expression, an expression vector of pPICZαA-HSA-CLP was constructed by the golden gate cloning strategy before being transformed into Pichia pastoris and integrated into the genome. The recombinant CLP was purified through the Ni-NTA affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. The Limulus amebocyte lysate (LAL) test exhibited that the hybrid peptide CLP inhibited lipopolysaccharides (LPS) in a dose-dependent manner and was significantly (p < 0.05) more efficient compared to the parent peptides. In addition, it essentially diminished (p < 0.05) the levels of nitric oxide and pro-inflammatory cytokines (including TNF-α, IL6, and IL-1β) in LPS-induced mouse RAW264.7 macrophages. As an attendant to the control and the parental peptide LL37, the number of LPS-induced apoptotic cells was diminished compared to the control parental peptide LL37 (p < 0.05) with the treatment of CLP. Consequently, we concluded that the hybrid peptide CLP might be used as a therapeutic agent.

Despite the development of various methods and commercial kits, site-directed mutagenesis of large plasmids remains a challenge in many laboratories. A site-directed mutagenesis method was developed for large plasmids by directly transforming two overlapping PCR fragments into  Escherichia coli . This method successfully generated mutations for plasmids of 8.3 kb and 11.0 kb with high efficiencies. The method only requires Q5 DNA polymerase and DpnI , which greatly reduces costs. The procedure is simple, including PCR reaction, DpnI treatment and transformation. This simple, efficient and economical site-directed mutagenesis method for large plasmids is likely to be widely applied in the future.

Dermatophytosis, an infectious disease caused by several fungi, can affect the hair, nails, and/or superficial layers of the skin and is of global significance. The most common dermatophytes in cats and dogs are Microsporum canis and Trichophyton mentagrophytes . Wood's lamp examination, microscopic identification, and fungal culture are the conventional clinical diagnostic methods, while PCR (Polymerase Chain Reaction) and qPCR (Quantitative PCR) are playing an increasingly important role in the identification of dermatophytes. However, none of these methods could be applied to point-of-care testing (POCT). The recent development of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic platform promises a rapid, accurate, and portable diagnostic tool. In this paper, we present a Cas12a-fluorescence assay to detect and differentiate the main dermatophytes in clinical samples with high specificity and sensitivity. The Cas12a-based assay was performed with a combination of recombinase polymerase amplification (RPA). The results could be directly visualized by naked eyes under blue light, and all tested samples were consistent with fungal culture and sequencing results. Compared with traditional methods, the RPA-Cas12a-fluorescence assay requires less time (about 30 min) and less complicated equipment, and the visual changes can be clearly observed with naked eyes, which is suitable for on-site clinical diagnosis.

Farnesoid X receptor (FXR) is a nuclear receptor known to markedly alter expression of major transporters and enzymes in the liver. However, its effects toward organic anion transporting polypeptides (OATP) 1B1 and 1B3 remain poorly characterized. Therefore, the present study was aimed at determining the effects of chenodeoxycholic acid (CDCA), a naturally occurring FXR agonist, on OATP1B expression in cynomolgus monkeys. Multiple administrations of 50 and 100 mg/kg of CDCA were first shown to significantly repress mRNA expression of SLCO1B1/3 approximately 60% to 80% in monkey livers. It also suppressed cytochrome P450 (CYP)7A1-mRNA and induced OST α / β -mRNA, which are well known targets of FXR and determinants of bile acid homeostasis. CDCA concomitantly decreased OATP1B protein abundance by approximately 60% in monkey liver. In contrast, multiple doses of 15 mg/kg rifampin (RIF), a pregnane X receptor agonist, had no effect on hepatic OATP1B protein, although it induced the intestinal P-glycoprotein and MR2 proteins by ∼2-fold. Moreover, multiple doses of CDCA resulted in a steady ∼2- to 10-fold increase of the OATP1B biomarkers coproporphyrins (CPs) in the plasma samples collected prior to each CDCA dose. Additionally, 3.4- to 11.2-fold increases of CPI and CPIII areas under the curve were observed after multiple administrations compared with the single dose and vehicle administration dosing groups. Taken together, these data suggest that CDCA represses the expression of OATP1B1 and OATP1B3 in monkeys. Further investigation of OATP1B downregulation by FXR in humans is warranted, as such downregulation effects may be involved in bile acid homeostasis and potential drug interactions in man. SIGNIFICANCE STATEMENT: Using gene expression and proteomics tools, as well as endogenous biomarker data, for the first time, we have demonstrated that OATP1B expression was suppressed and its activity was reduced in the cynomolgus monkeys following oral administration of 50 and 100 mg/kg/day of chenodeoxycholic acid (CDCA), a Farnesoid X receptor agonist, for 8 days. These results lead to a better understanding of OATP1B downregulation by CDCA and its role on bile acid and drug disposition., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Nrf2-mediated oxidative stress is a promising target of exhaustive exercise-induced fatigue (EEIF). Trilobatin (TLB) is a naturally occurring food additive with antioxidant effect and Nrf2 activation potency. The present study aimed to investigate the effect of TLB on EEIF and elucidate its underlying mechanism. Our results showed that TLB exerted potent anti-EEIF effect, as reflected by the rope climbing test and exhaustive swimming test. Moreover, TLB also effectively reduced the levels of lactate, creatine kinase, and blood urea nitrogen, and increased liver glycogen and skeletal muscle glycogen in mice after EEIF insult. Additionally, TLB also balanced the redox status as evidenced by decreasing the generation of reactive oxygen species and improving the antioxidant enzyme activities including superoxide dismutase, catalase, and glutathione peroxidase, as well as the level of glutathione both in the tissue of muscle and myocardium. Furthermore, TLB promoted nuclear factor erythroid 2-related factor 2 (Nrf2) from the cytoplasm to the nucleus, and upregulated its downstream antioxidant response element (ARE) including quinone oxidoreductase-1 and heme oxygenase-1. Intriguingly, TLB also upregulated the GPx4 protein expression and reduced iron overload in mice after EEIF insult. Encouragingly, the beneficial effect of TLB on EEIF-induced oxidative stress and ferroptosis were substantially abolished in Nrf2-deficient mice. In conclusion, our findings demonstrate, for the first time, that TLB alleviates EEIF-induced oxidative stress through mediating Nrf2/ARE/ferroptosis axis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xiao, Wei, Zhang, Xu, Ma, Gong, Gao and Xu.)

In an effort to control the outbreak of the African Swine Fever Virus (ASFV), there is an urgent need to develop an effective method to prevent the pandemic, including vaccines and diagnostic methods. The major capsid protein of ASFV p72 (B646L), which forms a trimer with each monomer adopting a double jelly roll fold, is the main component of the virus particle and major antigen of ASFV. Thus, the p72 protein may be considered an antigen candidate for vaccine and diagnostic development. However, the development of ASFV p72 trimer for the industry application, including veterinary usage, faces unavoidable challenges: firstly, the low cost of the antigen production is required in vaccine and diagnostic application; and, secondly, whether produced antigen folds in its native conformation. Here, based on the information provided by the atomic structure of p72, we have successfully performed rational mutagenesis on p72 trimers and expressed it in Saccharomyces cerevisiae with high yields. The cryo-EM structure of recombinant expressed p72 trimer is determined at 4.18 Å in resolution. The correlation coefficient between this structure and the ASFV virus structure is 0.77, suggesting a highly similar fold of this trimer with the native protein on the virus particle., Competing Interests: The authors declare a patient (CN 202110522777.4) issued for the preparation method and the application of African Swine Fever Virus capsid protein p72 trimer., (Copyright © 2022 Meng, Zhang, Liu, Huyan, Zhu, Xiang and Meng.)

Fungal infections are a major health concern because of limited antifungal drugs and development of drug resistance. Candida can develop azole drug resistance by overexpression of drug efflux pumps or mutating ERG11 , the target of azoles. However, the role of epigenetic histone modifications in azole-induced gene expression and drug resistance is poorly understood in Candida glabrata. In this study, we show that Set1 mediates histone H3K4 methylation in C. glabrata. In addition, loss of SET1 and histone H3K4 methylation increases azole susceptibility in both C. glabrata and S. cerevisiae. This increase in azole susceptibility in S. cerevisiae and C. glabrata strains lacking SET1 is due to distinct mechanisms. For S. cerevisiae, loss of SET1 decreased the expression and function of the efflux pump Pdr5, but not ERG11 expression under azole treatment. In contrast, loss of SET1 in C. glabrata does not alter expression or function of efflux pumps. However, RNA sequencing revealed that C. glabrata Set1 is necessary for azole-induced expression of all 12 genes in the late ergosterol biosynthesis pathway, including ERG11 and ERG3. Furthermore, chromatin immunoprecipitation analysis shows histone H3K4 trimethylation increases upon azole-induced ERG gene expression. In addition, high performance liquid chromatography analysis indicated Set1 is necessary for maintaining proper ergosterol levels under azole treatment. Clinical isolates lacking SET1 were also hypersusceptible to azoles which is attributed to reduced ERG11 expression but not defects in drug efflux. Overall, Set1 contributes to azole susceptibility in a species-specific manner by altering the expression and consequently disrupting pathways known for mediating drug resistance.

The purification of virus particles is an essential process for the manufacture of vaccines. However, the application of different purification processes may affect the quality of the virus particles, such as structural integrity and homogeneity, which may further influence the infectivity and immunogenicity of the purified virus. In this study, we took Feline calicivirus (FCV), a common natural pathogen in cats belonging to Caliciviridae , as a research model. By using cryo-electron microscopy (cryo-EM), we incorporated the 3D classification process as a virus flexibility evaluation system. Cryo-EM images of virus particles resulting from different purification processes were compared at near-atomic resolution. The results indicated that molecular sieving purification will impact the stability of P-domains through increasing flexibility as determined by the evaluation system, which can be extended to assess the purification effect on the entire particle. This evaluation process can be further applied to all non-enveloped viruses.

Background: The degree of human hepatocyte replacement in chimeric mice with humanized liver has previously been shown to correlate with human plasma albumin measurements. However, there are no reports that directly compare the remaining endogenous mouse albumin with the newly expressed human albumin following engraftment. To better understand the disposition of serum albumin in PXB-mice, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to simultaneously quantitate both human and mouse albumin from plasma. Results: A robust correlation was observed between the serum human albumin levels measured by LC-MS/MS and the estimated replacement index of PXB-mice. Conclusion: All data were shown to be specific and suitable to accurately quantify both human and mouse albumin from plasma of chimeric mice with humanized livers.

African swine fever is a widespread and highly contagious disease in the porcine population, which is caused by African swine fever virus (ASFV). The PCR and ELISA detection methods are the main conventional diagnostic methods for ASFV antigen/antibody detection in the field. However, these methods have limitations of expensive equipment, trained technicians, and time-consuming results. Thus, a rapid, inexpensive, accurate and on-site detection method is urgently needed. Here we describe a double-antigen-sandwich lateral-flow assay based on gold nanoparticle-conjugated ASFV major capsid protein p72, which can detect ASFV antibody in serum samples with high sensitivity and specificity in 10 min and the results can be determined by naked eyes. A lateral flow assay was established by using yeast-expressed and acid-treated ASFV p72 conjugated with gold nanoparticles, which are synthesized by seeding method. A high coincidence (97.8%) of the assay was determined using clinical serum compared to a commercial ELISA kit. In addition, our lateral flow strip can detect as far as 1:10,000 diluted clinically positive serum for demonstration of high sensitivity. In summary, the assay developed here was shown to be rapid, inexpensive, accurate and highly selective. It represents a reliable method for on-site ASFV antibody detection and may help to control the ASFV pandemic., Competing Interests: Outside of the submitted work, we hold a patent (CN 202110522777.4) issued for the preparation method and the application of African swine fever virus capsid protein p72 trimer., (Copyright © 2022 Zhu, Meng, Zhang, Bu, Zhao and Meng.)

As an expansion investigation of drug-drug interaction (DDI) from previous clinical trials, additional plasma endogenous metabolites were quantitated in the same subjects to further identify the potential biomarkers of organic anion transporter (OAT) 1/3 inhibition. In the single dose, open label, three-phase with fixed order of treatments study, 14 healthy human volunteers orally received 1000 mg probenecid alone, or 40 mg furosemide alone, or 40 mg furosemide at 1 hour after receiving 1000 mg probenecid on days 1, 8, and 15, respectively. Endogenous metabolites including kynurenic acid, xanthurenic acid, indo-3-acetic acid, pantothenic acid, p -cresol sulfate, and bile acids in the plasma were measured by liquid chromatography-tandem mass spectrometry. The C max of kynurenic acids was significantly increased about 3.3- and 3.7-fold over the baseline values at predose followed by the treatment of probenecid alone or in combination with furosemide respectively. In comparison with the furosemide-alone group, the C max and area under the plasma concentration-time curve (AUC) up to 12 hours of kynurenic acid were significantly increased about 2.4- and 2.5-fold by probenecid alone, and 2.7- and 2.9-fold by probenecid plus furosemide, respectively. The increases in C max and AUC of plasma kynurenic acid by probenecid are comparable to the increases of furosemide C max and AUC reported previously. Additionally, the plasma concentrations of xanthurenic acid, indo-3-acetic acid, pantothenic acid, and p -cresol sulfate, but not bile acids, were also significantly elevated by probenecid treatments. The magnitude of effect size analysis for known potential endogenous biomarkers demonstrated that kynurenic acid in the plasma offers promise as a superior addition for early DDI assessment involving OAT1/3 inhibition. SIGNIFICANCE STATEMENT: This article reports that probenecid, an organic anion transporter (OAT) 1 and OAT3 inhibitor, significantly increased the plasma concentrations of kynurenic acid and several uremic acids in human subjects. Of those, the increases of plasma kynurenic acid exposure are comparable to the increases of furosemide by OAT1/3 inhibition. Effect size analysis for known potential endogenous biomarkers revealed that plasma kynurenic acid is a superior addition for early drug-drug interaction assessment involving OAT1/3 inhibition., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Sr 2 Be 2 B 2 O 7 (SBBO) has long been considered as one of the most promising deep-ultraviolet nonlinear optical materials, but its crystal structure described by space group P 6̅ c 2 in previous studies has remained questionable. On the basis of first-principles calculations coupled with the high-throughput crystal structure prediction method, we found three energetically favorable structures for SBBO with space groups Cm , Pm , and P 6̅. These structures and a superstructure of space group Pm -S derived from the Cm structure were refined by the Rietveld method using the available powder X-ray diffraction data. These analyses show that the Pm -S structure is the best one, but its parent Cm structure is almost equally good and has the advantage of having higher symmetry. Via atom response theory analysis, we resolved the cause for the second-harmonic generation (SHG) responses of SBBO at the atomic and orbital level to elucidate the importance of local inversion symmetry in reducing the SHG response.

The development of a quick, single-step cloning system for generation of multiexon gene expression constructs is presented. The system allows efficient and cost-effective assembly of multiple exons of interest genes into different expression plasmids in both Escherichia coli and Pichia pastoris . The high cloning efficiency and low cost of the system make it ideal for a novel workflow for the assembly of intron-bearing genes for expression in two different expression hosts.

African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF., Competing Interests: The authors have declared that no competing interests exist.

Targeted genome disruptions and single-nucleotide conversions with the CRISPR/Cas system have greatly facilitated the development of gene therapy, basic biological research, and synthetic biology. With vast progress in this field, there are still aspects to be optimized, including the target range, the ability to multiplex, the mutation efficiency and specificity, as well as the requirement of adjusting protospacer adjacent motifs (PAMs). Here, we report the development of a highly efficient genome disruption and single-nucleotide conversion tool with a gRNA-tRNA array and SpCas9-NG (GTR 2.0). We performed gene disruptions in yeast cells covering all 16 possible NGN PAMs and all 12 possible single-nucleotide conversions (N to N) with near 100% efficiencies. Moreover, we applied GTR 2.0 for multiplexed single-nucleotide conversions, resulting in 66.67% mutation efficiency in simultaneous generation of 4 single-nucleotide conversions in one gene, as well as 100% mutation efficiency for simultaneously generating 2 single-nucleotide conversions in two different genes. GTR 2.0 will substantially expand the scope, efficiency, and capabilities of yeast genome editing, and will be a versatile and invaluable addition to the toolbox of synthetic biology and metabolic engineering.

Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals. However, as a non-oleaginous yeast, S. cerevisiae has a limited production capacity for lipophilic compounds, such as β-carotene. To increase its accumulation of β-carotene, we engineered different lipid metabolic pathways in a β-carotene producing strain and investigated the relationship between lipid components and the accumulation of β-carotene. We found that overexpression of sterol ester synthesis genes ARE1 and ARE2 increased β-carotene yield by 1.5-fold. Deletion of phosphatidate phosphatase (PAP) genes (PAH1, DPP1, and LPP1) also increased β-carotene yield by twofold. Combining these two strategies resulted in a 2.4-fold improvement in β-carotene production compared with the starting strain. These results demonstrated that regulating lipid metabolism pathways is important for β-carotene accumulation in S. cerevisiae, and may also shed insights to the accumulation of other lipophilic compounds in yeast., (© 2021 Wiley Periodicals LLC.)

Objectives: Advances in genetic technologies provide opportunities for patient care and ethical challenges. Clinical care of patients with rare Mendelian disorders is often at the forefront of those developments. Whereas in classical polygenic inflammatory bowel disease (IBD), the predictive value of genetic variants is very low, predictive prenatal genetic diagnosis can inform families at high risk of severe genetic disorders. Patients with IL-10 signalling defects because of pathogenic variants in IL10RA, Il10RB, and IL10 develop severe infantile onset inflammatory bowel disease that is completely penetrant and has a high morbidity and substantial mortality despite treatment., Methods: We performed a survey among tertiary specialist paediatric centers of 10 countries on the utilization of predictive prenatal genetic diagnosis in IL-10 signalling defects. We retrospectively report prenatal genetics in a series of 8 families., Results: International variation in legislation, guidelines, expert opinion, as well as cultural and religious background of families and clinicians results in variable utilization of preimplantation and prenatal genetic testing for IL-10 signalling defects. Eleven referrals for prenatal diagnosis for IL-10 signalling defects were identified across 4 countries. We report on 8 families who underwent prenatal preimplantation monogenic testing after in vitro fertilization (n = 2) and/or by amniocentesis/chorion villus sampling (n = 6). A genetic diagnosis was established in 1 foetus and excluded in 7 foetuses (all IL10RA variants)., Conclusions: Prenatal genetic testing for IL10R-defects is feasible, yet the legal and ethical considerations are complex and controversial. In some countries, predictive genetics for IL-10-related signalling defects is entering clinical practice., Competing Interests: The authors report no conflict of interest., (Copyright © 2020 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Expression and functional changes in the organic anion transporting polypeptide (OATP)-multidrug resistance-associated protein (MRP) axis of transporters are well reported in patients with nonalcoholic steatohepatitis (NASH). These changes can impact plasma and tissue disposition of endo- and exogenous compounds. The transporter alterations are often assessed by administration of a xenobiotic or by transporter proteomic analysis from liver biopsies. Using gene expression, proteomics, and endogenous biomarkers, we show that the gene expression and activity of OATP and MRP transporters are associated with disease progression and recovery in humans and in preclinical animal models of NASH. Decreased OATP and increased MRP3/4 gene expression in two cohorts of patients with steatosis and NASH, as well as gene and protein expression in multiple NASH rodent models, have been established. Coproporphyrin I and III (CP I and III) were established as substrates of MRP4. CP I plasma concentration increased significantly in four animal models of NASH, indicating the transporter changes. Up to a 60-fold increase in CP I plasma concentration was observed in the mouse bile duct-ligated model compared with sham controls. In the choline-deficient amino acid-defined high-fat diet (CDAHFD) model, CP I plasma concentrations increased by >3-fold compared with chow diet-fed mice. In contrast, CP III plasma concentrations remain unaltered in the CDAHFD model, although they increased in the other three NASH models. These results suggest that tracking CP I plasma concentrations can provide transporter modulation information at a functional level in NASH animal models and in patients. SIGNIFICANCE STATEMENT: Our analysis demonstrates that multidrug resistance-associated protein 4 (MRP4) transporter gene expression tracks with nonalcoholic steatohepatitis (NASH) progression and intervention in patients. Additionally, we show that coproporphyrin I and III (CP I and III) are substrates of MRP4. CP I plasma and liver concentrations increase in different diet- and surgery-induced rodent NASH models, likely explained by both gene- and protein-level changes in transporters. CP I and III are therefore potential plasma-based biomarkers that can track NASH progression in preclinical models and in humans., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Manure amendment generally bolsters soil organisms but not all bacteria equally. To understand why different taxa respond differently, we used shotgun metagenomic approaches to profile functional potentials and correlate them with taxon abundances. A soil originally unproductive was reclaimed using commercial manure and finally became productive. The abundance of Firmicutes in the soil decreased, whereas that of Bacteroidetes and Proteobacteria increased after manure addition. Thirty-nine KEGG modules were significantly different across fertilizer treatments. These modules were mainly associated with the phosphoenolpyruvate-dependent phosphotransferase system (PTS), ATP-binding cassette (ABC) transporters, and two-component signal transduction systems. The Proteobacteria and Firmicutes mainly contributed to these modules. Correlation between the abundances of phyla and orthologs showed two distinctive patterns. One linked the Firmicutes to cell wall biosynthesis, PTS, and ABC transporters, and the other linked the Betaproteobacteria , Bacteroidetes , and Verrucomicrobia to lipopolysaccharide biosynthesis, bacterial motility, and carbon metabolism. Correlation between the abundances of phyla and Carbohydrate-Active Enzyme Database families also showed two distinctive patterns, one of them linking the Betaproteobacteria , Bacteroidetes , and Verrucomicrobia to very high abundances of glycosyltransferases and glycoside hydrolases. Overall, the Proteobacteria and Firmicutes were main drivers of functional potential differences across fertilizer treatments. The Firmicutes were enriched with genes associated with cell wall biosynthesis and membrane transports, while Proteobacteria with lipopolysaccharide biosynthesis and carbohydrate metabolism, which supports our hypothesis that the Firmicutes have a lower potential for utilizing manure-derived carbohydrates, while Proteobacteria have a higher potential. This explains why the Proteobacteria and Firmicutes responded to manure differently.

Plasma pyridoxic acid (PDA) and homovanillic acid (HVA) were recently identified as novel endogenous biomarkers of organic anion transporter (OAT) 1/3 function in monkeys. Consequently, this clinical study assessed the dynamic changes and utility of plasma PDA and HVA as an initial evaluation of OAT1/3 inhibition in early-phase drug development. The study was designed as a single-dose randomized, three-phase, crossover study; 14 Indian healthy volunteers received probenecid (PROB) (1000 mg orally) alone, furosemide (FSM) (40 mg orally) alone, or FSM 1 hour after receiving PROB (40 and 1000 mg orally) on days 1, 8, and 15, respectively. PDA and HVA plasma concentrations remained stable over time in the prestudy and FSM groups. Administration of PROB significantly increased the area under the plasma concentration-time curve (AUC) of PDA by 3.1-fold (dosed alone; P < 0.05), and 3.2-fold (coadministered with FSM; P < 0.01), compared with the prestudy and FSM groups, respectively. The corresponding increase in HVA AUC was 1.8-fold ( P > 0.05) and 2.1-fold ( P < 0.05), respectively. The increases in PDA AUC are similar to those in FSM AUC, whereas those of HVA are smaller (3.1-3.2 and 1.8-2.1 vs. 3.3, respectively). PDA and HVA renal clearance ( CL R ) values were decreased by PROB to smaller extents compared with FSM (0.35-0.37 and 0.67-0.73 vs. 0.23, respectively). These data demonstrate that plasma PDA is a promising endogenous biomarker for OAT1/3 function and that its plasma exposure responds in a similar fashion to FSM upon OAT1/3 inhibition by PROB. The magnitude and variability of response in PDA AUC and CL R values between subjects is more favorable relative to HVA., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Objective: To report on prenatal diagnosis and follow up of two patients with paternal uniparental disomy of chromosome 6 (pUPD6)., Methods: Fetal cells were subjected to in situ culturing and G-banded chromosomal analysis. DNA samples of the fetuses and their parents were also analyzed with single nucleotide polymorphism microarray (SNP array)., Results: Both fetuses had a normal male karyotype. SNP array analysis showed both have carried pUPD6., Conclusion: pUPD6 can lead to transient neonatal diabetes mellitus type 1. Homozygous status of recessive mutations, disorder of gene imprinting, and its influence on placental function are the main factors to be considered during prenatal diagnosis and genetic counseling for pUPD6.

Aim: This study aimed to study the effects of intratracheal instillation of budesonide on lung maturity of premature fetal rabbits. The developmental pattern of pulmonary alveoli in rabbits is similar to that in humans., Method: Fetal rabbits were taken out from female rabbits on the 28th day of pregnancy (full term = 31 days) by cesarean section (c-section). The fetal rabbits were divided into four groups: control (normal saline, NS), budesonide (budesonide, BUD), calf pulmonary surfactant for injection (pulmonary surfactant, PS), and calf pulmonary surfactant + budesonide for injection (pulmonary surfactant + budesonide, PS + BUD). All premature rabbits were kept warm after c-section. After 15-min autonomous respiration, a tracheal cannula was implemented for instilling NS, BUD, PS, and PS + BUD. The morphology of lung tissues of premature fetal rabbits was analyzed using optical and electron microscopes. Surfactant protein B (SP-B) mRNA and protein levels in lung tissues were determined using polymerase chain reaction and Western blotting, respectively., Result: Intratracheal instillation of BUD could increase the alveolar area of the fetal rabbits (P < 0.01), decrease the alveolar wall thickness (P < 0.01), and increase the mean density of lamellar bodies (P < 0.05) and SP-B protein levels in type II epithelial cells of pulmonary alveoli (P < 0.05)., Conclusion: Intratracheal instillation of BUD during early postnatal life is effective in promoting alveolarization and increasing SP-B expression, the pro-pulmonary maturity of BUD combined with PS is superior to that of BUD or PS alone. However, the long-term effect of BUD on lung development needs further exploration., (© 2017 Wiley Periodicals, Inc.)

Sex chromosomes evolved from autosomes many times across the eukaryote phylogeny. Several models have been proposed to explain this transition, some involving male and female sterility mutations linked in a region of suppressed recombination between X and Y (or Z/W, U/V) chromosomes. Comparative and experimental analysis of a reference genome assembly for a double haploid YY male garden asparagus (Asparagus officinalis L.) individual implicates separate but linked genes as responsible for sex determination. Dioecy has evolved recently within Asparagus and sex chromosomes are cytogenetically identical with the Y, harboring a megabase segment that is missing from the X. We show that deletion of this entire region results in a male-to-female conversion, whereas loss of a single suppressor of female development drives male-to-hermaphrodite conversion. A single copy anther-specific gene with a male sterile Arabidopsis knockout phenotype is also in the Y-specific region, supporting a two-gene model for sex chromosome evolution.

In the present investigations, we evaluate in vitro hepatocyte uptake and partitioning for the prediction of in vivo clearance and liver partitioning. Monkeys were intravenously co-dosed with rosuvastatin and bosentan, substrates of the organic anion transporting polypeptides (OATPs), and metformin, a substrate of organic cation transporter 1 (OCT1). Serial plasma and liver samples were collected over time. Liver and plasma unbound fraction was determined using equilibrium dialysis. In vivo unbound partitioning (Kp u,u ) for rosuvastatin, bosentan, and metformin, calculated from total concentrations in the liver and plasma, were 243, 553, and 15, respectively. A physiologically based pharmacokinetic monkey model that incorporates active and passive hepatic uptake was developed to fit plasma and liver concentrations. In addition, a two-compartment model was used to fit in vitro hepatic uptake curves in suspended monkey hepatocyte to determine active uptake, passive diffusion, and intracellular unbound fraction parameters. At steady-state in the model, in vitro Kp u,u was determined. The results demonstrated that in vitro values under-predicted in vivo active uptake for rosuvastatin, bosentan, and metformin by 6.7-, 28-, and 1.5-fold, respectively, while passive diffusion was over-predicted. In vivo Kp u,u values were under-predicted from in vitro data by 30-, 79-, and 3-fold. In conclusion, active uptake and liver partitioning in monkeys for OATP substrates were greatly under-predicted from in vitro hepatocyte uptake, while OCT-mediated uptake and partitioning scaled reasonably well from in vitro, demonstrating substrate- and transporter-dependent scaling factors. The combination of in vitro experimental and modeling approaches proved useful for assessing prediction of in vivo intracellular partitioning.

The garden asparagus stem blight caused by filamentous fungus Phomopsis asparagi exposes a serious threat on asparagus production globally. However, to present, we understand poorly about the molecular mechanisms of fungal pathogenicity. To facilitate functional genomics research of P. asparagi, here we developed a highly efficient and stable Agrobacterium tumefaciens-mediated transformation approach which yielded 150-200 transformants per 1 × 10 6 conidia. Our results indicated that 25 °C, acetosyringone concentration of 150 μmol/L, and 72 h were recommended as optimal co-cultivation conditions for the transformation. Using this transformation approach, we constructed a T-DNA insertion mutant library containing 1253 strains. Twenty randomly selected T-DNA insertion mutants were able to grow on 0.2 × PDA selective media after five successive subcultures without selective pressure, indicating that the exogenous T-DNA was stably integrated into the P. asparagi genome. We confirmed several randomly selected mutants using PCR with primers specific to the hph gene. Southern blots suggested that three out of the five selected mutants have a single T-DNA insertion. Interestingly, multiple mutant candidates with growth defects were obtained from the growth assay. Moreover, several mutants were selected for further analysis on the T-DNA flanking sequences through TAIL-PCR analysis. A sequence comparison of total junction fragments implied that the insertion of T-DNA within P. asparagi genome appeared to be a random event. The transformation technology and genetic resources developed here will facilitate studies of pathogenic mechanisms in this devastating filamentous fungal pathogen of garden asparagus.

Objectives: The purpose of this study was to evaluate the diagnostic utility of karyotype analysis of amniotic fluid for fetuses with abnormal sonographic findings and to determine the detection rates of abnormal karyotypes., Methods: We conducted a retrospective study of 5328 fetuses with abnormal sonographic findings in the first or second trimester enrolled from October 1998 and September 2015. Cytogenetic results from amniotic fluid were obtained in all of these pregnancies. Sonographic abnormalities were stratified according to anatomic system involvement., Results: A total of 238 abnormal karyotypes were encountered in the 5328 fetuses (4.5%). The highest rate of chromosomal anomalies was in fetuses with structural abnormalities in multiple organ systems (25.7%), followed by an abnormal amniotic fluid volume (7.9%), structural abnormalities in a single system (7.3%), multiple nonstructural anomalies (7.2%), isolated placental abnormalities (7.1%), and isolated soft markers for aneuploidy (2.4%; P < .01). Among abnormalities in a single system, gastrointestinal and neck/body fluids had particularly high detection rates (26.1% and 26.2%, respectively). A detailed analysis suggested that the probability of an abnormal karyotype among every anatomic system was statistically significant (P < .01). This study identified several common indications with extremely high abnormal rates: duodenal atresia (53.1%), holoprosencephaly (48.8%), fetal hydrops (39.5%), cerebellar hypoplasia (32.0%), cystic hygroma (31.5%), absent/short nasal bone (11.0%), and bilateral choroid plexus cysts (8.5%)., Conclusions: Cytogenetic analysis has important clinical utility in a wide range of settings, such as prenatal diagnosis. For fetuses with indications of a highly abnormal detection rate, karyotype analysis should be suggested., (© 2017 by the American Institute of Ultrasound in Medicine.)

The humanized liver mouse model is being exploited increasingly for human drug metabolism studies. However, its model stability, intercommunication between human hepatocytes and mouse nonparenchymal cells in liver and murine intestine, and changes in extrahepatic transporter and enzyme expressions have not been investigated. We examined these issues in FRGN [fumarylacetoacetate hydrolase (Fah-/-), recombination activating gene 2 (Rag2-/-), and interleukin 2 receptor subunit gamma (IL-2rg -/-) triple knockout] on nonobese diabetic (NOD) background] and chimeric mice: mFRGN and hFRGN (repopulated with mouse or human hepatocytes, respectively). hFRGN mice showed markedly higher levels of liver cholesterol, biliary bilirubin, and bile acids (liver, bile, and plasma; mainly human forms, but also murine bile acids) but lower transforming growth factor beta receptor 2 (TGFBR2) mRNA expression levels (10%) in human hepatocytes and other proliferative markers in mouse nonparenchymal cells (Tgf-β1) and cholangiocytes [plasma membrane-bound, G protein-coupled receptor for bile acids (Tgr5)], suggestive of irregular regeneration processes in hFRGN livers. Changes in gene expression in murine intestine, kidney, and brain of hFRGN mice, in particular, induction of intestinal farnesoid X receptor (Fxr) genes: fibroblast growth factor 15 (Fgf15), mouse ileal bile acid binding protein (Ibabp), small heterodimer partner (Shp), and the organic solute transporter alpha (Ostα), were observed. Proteomics revealed persistence of remnant murine proteins (cyotchrome P450 7α-hydroxylase (Cyp7a1) and other enzymes and transporters) in hFRGN livers and suggest the likelihood of mouse activity. When compared with normal human liver tissue, hFRGN livers showed lower SHP mRNA and higher CYP7A1 (300%) protein expression, consequences of tβ- and tα-muricholic acid-mediated inhibition of the FXR-SHP cascade and miscommunication between intestinal Fgf15 and human liver fibroblast growth factor receptor 4 (FGFR4), as confirmed by the unchanged hepatic pERK/total ERK ratio. Dysregulation of hepatocyte proliferation and bile acid homeostasis in hFRGN livers led to hepatotoxicity, gallbladder distension, liver deformity, and other extrahepatic changes, making questionable the use of the preparation for drug metabolism studies., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Down syndrome is caused by partial or complete triplication of genes located on chromosome 21. Its incidence increases dramatically with the age of women. Hypotheses proposed for this have included abnormal homologous recombination, defective spindle assembly, biological aging, reduction of cohesion complexes, endocrine disorders, oocyte selection model, and single nucleotide polymorphisms of genes that maintain chromosome stability, etc. A literature review is provided here.

The effect of quadrivalent geometry on meiotic behaviour was evaluated. Segregation patterns of 404 cleavage stage embryos from 40 reciprocal translocation carriers undergoing 75 PGD cycles were analysed according to the asymmetric degree of quadrivalent. The percentage of alternate products with severe asymmetric quadrivalents was significantly lower than patients with mild asymmetric quadrivalents (22.5% versus 38.7%, P = 0.001). The incidence of 3:1 products was significantly higher in patients with severe compared with mild asymmetric quadrivalents (23.1% versus 12.2%, P = 0.004). The incidence of adjacent 1 (25.8% versus 24.3%), 2 (11.5% versus 12.6%) and 4:0/other segregation products (17.0% versus 12.2%) were not statistically significantly different between embryos from patients with severe or mild asymmetric quadrivalents. After adjusting for the confounder of sex using a logistic regression model, the odds of alternate embryos is about one-half for carriers classified as severe (OR 0.456, 95% CI 0.291 to 0.705), and the odds of 3:1 embryos is 2.2 times higher for carriers with severe asymmetric quadrivalents (OR 2.235, 95% CI 1.318 to 3.846). Our results suggest that the meiotic segregation pattern is related to the degree of asymmetry of specific quadrivalents. Severe asymmetric quadrivalents increases the risk of abnormal embryos., (Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2 kinases with IC50 values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft model.