Use of precision cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the 32P-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene

In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the ³²P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 μM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6β-OH-androstendione, 6β-OH-TES, and 15β-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10⁹ nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.     

Influence of carbon sources on the viability and resuscitation of Acetobacter senegalensis during high-temperature gluconic acid fermentation

Much research has been conducted about different types of fermentation at high temperature, but only a few of them have studied cell viability changes during high-temperature fermentation. In this study, Acetobacter senegalensis, a thermo-tolerant strain, was used for gluconic acid production at 38 °C. The influences of different carbon sources and physicochemical conditions on cell viability and the resuscitation of viable but nonculturable (VBNC) cells formed during fermentation were studied. Based on the obtained results, A. senegalensis could oxidize 95 g l^− 1 glucose to gluconate at 38 °C (pH 5.5, yield 83%). However, despite the availability of carbon and nitrogen sources, the specific rates of glucose consumption (q_s) and gluconate production (q_p) reduced progressively. Interestingly, gradual q_s and q_p reduction coincided with gradual decrease in cellular dehydrogenase activity, cell envelope integrity, and cell culturability as well as with the formation of VBNC cells. Entry of cells into VBNC state during stationary phase partly stemmed from high fermentation temperature and long-term oxidation of glucose, because just about 48% of VBNC cells formed during stationary phase were resuscitated by supplementing the culture medium with an alternative favorite carbon source (low concentration of ethanol) and/or reducing incubation temperature to 30 °C. This indicates that ethanol, as a favorable carbon source, supports the repair of stressed cells. Since formation of VBNC cells is often inevitable during high-temperature fermentation, using an alternative carbon source together with changing physicochemical conditions may enable the resuscitation of VBNC cells and their use for several production cycles.

     

NMR spectroscopy-based metabolomic study of serum in sulfur mustard exposed patients with lung disease

Sulfur mustard (SM) is a vesication chemical warfare agent for which there is currently no antidote. Despite years of research, there is no common consensus about the pathophysiological basis of chronic pulmonary disease caused by this chemical warfare agent. In this study, we combined chemometric techniques with nuclear magnetic resonance (NMR) spectroscopy to explore the metabolic profile of sera from SM-exposed patients. A total of 29 serum samples obtained from 17 SM-injured patients, and 12 healthy controls were analyzed by Random Forest. Increased concentrations of seven amino acids, glycerol, dimethylamine, ketone bodies, lactate, acetate, citrulline and creatine together with the decreased very low-density lipoproteins (VLDL) levels were observed in patients compared with control subjects. Our study reveals the metabolic profile of sera from SM-injured patients and indicates that NMR-based methods can distinguish these patients from healthy controls.     

THE BIOTRANSFORMATION,BIODEGRADATION, AND BIOREMEDIATION OF ORGANIC COMPOUNDS BY MICROALGAE1

Rapid growth in the biotechnological industry and production has put tremendous pressure on the biological methods that may be used according to the guidelines of green chemistry. However, despite continuing dramatic increases in published research on organic biotransformation by microorganisms, more research exists with microalgae. Our efforts in transforming chemicals such as organic compounds for the production of functionalized products help to lessen the environmental effects of organic synthesis. These biotransformations convert organic contaminants to obtain carbon or energy for growth or as cosubstrates. This review aims to focus on the potential of microalgae in transformation, conversion, remediation, accumulation, degradation, and synthesis of various organic compounds. However, these technologies have the ability to provide the most efficient and environmentally safe approach for inexpensive biotransforming of a variety of organic contaminants, which are most industrial residues. In addition, the recent advances in microalgal bioactivity were discussed.     

Genetic variation in RPOIILS gene encoding RNA polymerase II largest subunit from Leishmania major

Leishmaniasis is a geographically widespread severe disease which includes visceral leishmaniasis, cutaneous leishmaniasis (CL). There are 350 million people at risk in over 80 countries. In the Old World, CL is usually caused by Leishmania major, Leishmania tropica, and Leishmania aethiopica complex which 90 % of cases occurring in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil, and Peru. Recently, some reports showed that some strains of L. major have internal transcribed space (ITS-1) with differential size exhibiting homology with the related gene in a divergent genus of kinetoplastida, the Crithidia. This prompted us to analyze the mentioned gene in 100 isolates obtained from patients with suspected CL. After obtaining samples from 100 patients, DNA extraction was performed and ITS-1 was analyzed using PCR–RFLP. These samples were sequenced for verifying their homology. Then, RPOIILS gene was analyzed in the samples that their ITS-1 gene exhibiting homology with the related gene in Crithidia. Results showed that 10 % of the isolates have ITS-1 exhibiting different size with the routine ones. Sequencing of them showed their similarity to the one from Crithidia fasciculata. RPOIILS gene encoding RNA polymerase II largest subunit analysis showed genetic diversity. This study might also help in solving the problems concerning Leishmaniasis outbreak currently facing in Iran and some other endemic regions of the world.     

FGF21 treatment ameliorates alcoholic fatty liver through activation of AMPK-SIRT1 pathway

Fibroblast growth factor 21 （FGF21）, a recently identified member of the FGF superfamily, is mainly secreted from the liver and adipose tissues and plays an important role in improving metabolic syndrome and homeostasis. The aim of this study is to evaluate the role of FGF21 in alcoholic fatty liver disease （AFLD） and to determine if it has a therapeutic effect on AFLD. In this paper, we tested the effect of FGF21 on alcohol-induced liver injury in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. Male KM mice received single dose of 5 g/kg ethanol gavage every day for 6 weeks, which induced sig- nificant fatty liver and liver injury. The alcohol-induced fatty liver cell model was achieved by adding ethanol into the medium of HepG2 cell cultures at a final concentration of 75 mM for 9 days. Results showed that treatment with recombinant FGF21 ameliorated alcoholic fatty liver and liver injury both in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. In addition, FGF21 treatment down-regulated the hepatic expression of fatty acid synthetic key enzyme, activated hepatic AMPK- SIRT1 pathway and significantly down-regulated hepatic oxidative stress protein. Taken together, FGF21 corrects multiple metabolic parameters of AFLD in vitro and in vivo by activation of the AMPK-SIRT1 pathway.     

Instantaneous wave-free ratio and fractional flow reserve in clinical practice

Objectives

To compare fractional flow reserve (FFR) and instantaneous wave-free ratio (iFR) measurements in an all-comer patient population with moderate coronary artery stenoses.

Background

Visual assessment of the severity of coronary artery stenoses is often discordant in moderate lesions. FFR allows reliable functional severity assessment in these cases but requires adenosine-induced hyperaemia with associated additional time, costs and side effects. The iFR is a hyperaemia-independent index.

Methods and results

Between November 2015 and February 2017, 356 consecutive patients were included in whom 515 coronary stenoses were measured using both iFR and FFR. Mean iFR and FFR were 0.90?±?0.09 and 0.86?±?0.08, respectively. iFR correlated well with FFR [r?=?0.75; p?<?0.001]. Receiver operating characteristic analysis identified an area under the curve of 0.92. An iFR-only strategy with a treatment cut-off ≤0.89 revealed a diagnostic classification agreement with the FFR-only strategy in 420 lesions (82%) with a sensitivity of 87%, a specificity of 80%, a positive predictive value of 56% and a negative predictive value of 96%.

Conclusions

Real-time iFR measurements have good negative predictive value compared to FFR, but moderate diagnostic accuracy (82%). It exposes fewer patients to adenosine, reduces procedure time and costs. Further prospective trials are needed to evaluate specific clinical settings, cut-off values and endpoints.