Development of Time-Resolved Fluorescence Immunochromatographic Assays for Simultaneously Detecting Tylosin and Tilmicosin in Milk in Group-Screening Manner

Tylosin and tilmicosin (T&T) residues in livestock products have received extensive attention from consumers. Time-resolved fluorescence immunochromatographic assay (TRFICA), as a fast, efficient and sensitive immunoassay method, has played an increasingly important role in the food safety field. Therefore, herein a quantitative and visual TRFICA was established for simultaneously detecting T&T in milk in a group-screening manner. Under the optimal conditions, the standard curve range of developed TRFICA based on the T&T was 1.87~7.47 ng/mL, and the half-maximal inhibition concentrations (IC50) were 4.06 ng/mL and 3.74 ng/mL, respectively. The limits of detection (LOD) of the TRFICA method were from 1.72 ng/mL to 1.39 ng/mL, and the visual cut-off values were 31.25 ng/mL and 62.50 ng/mL for T&T in milk, respectively. Moreover, the stability experiments showed that the strips could be stored at 4 °C for more than 6 months, the total detection time was less than 13 min, and the cross-reactivities (CRs) with related compounds were less than 0.1%, which concluded that the developed TRFICA method could be used in real milk sample detection.

A Pan-Inhibitor for Protein Arginine Methyltransferase Family Enzymes

Protein arginine methyltransferases (PRMTs) play important roles in transcription, splicing, DNA damage repair, RNA biology, and cellular metabolism. Thus, PRMTs have been attractive targets for various diseases. In this study, we reported the design and synthesis of a potent pan-inhibitor for PRMTs that tethers a thioadenosine and various substituted guanidino groups through a propyl linker. Compound II757 exhibits a half-maximal inhibition concentration (IC50) value of 5 to 555 nM for eight tested PRMTs, with the highest inhibition for PRMT4 (IC50 = 5 nM). The kinetic study demonstrated that II757 competitively binds at the SAM binding site of PRMT1. Notably, II757 is selective for PRMTs over a panel of other methyltransferases, which can serve as a general probe for PRMTs and a lead for further optimization to increase the selectivity for individual PRMT.

An Optimized Fluorometric Method to Test the Capacities of Cranberry Polyphenols and Metabolites to Inhibit the Adhesion of Type-P and Type-1 Fimbriated Uropathogenic E. coli

Objectives Adhesion of type-P and type-1 fimbriated uropathogenic E. coli to urinary tract epithelial cells initiates urinary tract infections. This research aimed to optimize and apply a fluorometric method to evaluate the capacities of cranberry polyphenols and metabolites to inhibit such adhesion in vitro. Methods BacLight Green labelled E. coli were incubated with cranberry polyphenols or microbial metabolites of cranberry polyphenols for 30 min at 37°C. Mixture was added to a 96-well microplate containing 1 × 105/well of human uroepithelial T24 cells and incubated for 1 h at 37°C. After incubation, E. coli not adhered were removed by phosphate buffer washing. Fluorescent intensity was measured on a microplate reader at 480 nm excitation and 516 nm emission. Results Stable and strong fluorescent readings were obtained with 800 μmol/L BacLight Green for E. coli labeling and an E. coli to T24 cells ratio of 400:1 for co-incubation. A standard curve was established using 0–63 μM myricetin. The half-maximal inhibitory concentrations (IC50) of myricetin were 13.2 μM against type-P E. coli adhesion and 5.5 μM against type-1 E. coli adhesion. A fraction enriched with procyanidin polymers had IC50 of 57.6 μg/mL against type-P E. coli and 19.3 μg/mL against type-1 E. coli, respectively. Its anti-adhesion activities were more potent than those of cranberry fractions enriched with procyanidin oligomers, flavonols, or anthocyanin. Procyanidin A2 had a maximal inhibition about 35% at 17.3 μM against type-P E. coli, but no anti-adhesion activity was observed against type-1 E. coli. Procyanidin B2 showed a plateaued inhibition about 15% at 173–691 μM against type-P E. coli. Its maximal inhibition against type-1 E. coli was around 25% at 346 μM. Hippuric acid, a major metabolite of cranberry polyphenols, had a maximal inhibition about 20% at 558 μM against type-1 E. coli adhesion, whereas its anti-adhesion activity against type-P E. coli was not detected. Conclusions The optimized fluorometric method showed that both structure and composition of cranberry polyphenols and metabolites affected their abilities to inhibit E. coli adhesion in vitro. Anti-adhesion activities of cranberry polyphenols also depend on type of E. coli fimbriae. Funding Sources University of Florida Research Foundation Seed Fund.

Results of a phase 1 study of SRF388, a first-in-human, first-in-class, high-affinity anti-IL-27 antibody in advanced solid tumors.

2551 Background: IL-27 is an immunosuppressive cytokine, consisting of two subunits p28 and EBI3, that upregulates immune checkpoint receptors (eg, PD-L1, TIGIT) and downregulates proinflammatory cytokines such as IFNγ, TNFα, and IL-17. SRF388 is a first-in-class, fully human IgG1 antibody to IL-27 that blocks the interaction between IL-27 and its receptor, thereby promoting immune activation in the tumor microenvironment. The IL-27 pathway is activated in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC), and high circulating levels of EBI3 are associated with inferior outcomes in both. Circulating EBI3 levels may serve as a predictive biomarker of SRF388 activity. Methods: Patients with advanced solid tumors refractory to standard therapy were enrolled in a phase 1 dose-escalation study (accelerated single patient followed by standard 3+3) to establish the preliminary safety of SRF388 as a monotherapy and to identify a dose suitable for expansion (NCT04374877). SRF388 was administered intravenously every 4 weeks. Tumor response was assessed by RECIST v1.1. SRF388 pharmacokinetic (PK) and pharmacodynamic (PD) [phospho-STAT (pSTAT) inhibition] analyses were performed. Results: As of January 26, 2021, 12 patients have received SRF388 at doses ranging from 0.003 to 10 mg/kg with 2 patients undergoing intra-patient dose escalation. Median age was 68 years, 67% were female, and ECOG PS was 0/1 (42%/58%). Median number of prior therapies was 2 (range 1–9), and 75% were anti-PD-(L)1 experienced (n = 9). The only treatment-related adverse events observed across dose levels were low-grade fatigue (n = 1, 8%), nausea (n = 1, 8%) and excess salivation (n = 1, 8%). No dose-limiting toxicities (DLTs) or ≥ Grade 3 related toxicity have occurred. Mean time on study is 12.5 weeks (range 4–40). One patient with RCC who received prior anti-PD-1 has prolonged stable disease for > 9 months. SRF388 PK are linear with estimated T1/2 ranging from 6–19 days. There is evidence of accumulation and no anti-drug antibody development to date. Maximal inhibition of the IL-27 signaling pathway as measured by > 90% pSTAT inhibition in whole blood was achieved starting at 0.3 mg/kg. Given combined evidence of near-complete pathway inhibition and preclinical human equivalent dose modeling projecting biologically active doses, additional slots were opened for RCC and HCC starting at 1 mg/kg. Conclusions: Preliminary results of IL-27 pathway blockade with a first-in-class therapeutic demonstrates that SRF388 is well tolerated at doses that achieve maximal inhibition of downstream pSTAT signaling through the dosing period. Expansions are planned in HCC and RCC. Updated data including the recommended phase 2 dose, clinical outcomes, PK/PD and correlative analyses will be presented. Clinical trial information: NCT04374877.

Effect of different carbon and nitrogen sources combination in medium for production of biocontrol agent Trichoderma harzianum

The increasing usage of chemicals for plant protection in recent years has become a serious problem. One of the possible solutions is use of beneficial microorganisms instead of synthetic fungicides, which will contribute to the protection of the environment and human health. Since the fungi Aspergillus flavus and Fusarium graminearum are the most important pathogens that cause maize diseases and produce mycotoxins, the potential of Trichoderma harzianum for biocontrol of both phytopathogens was examined in this paper. The aim of this paper was to study the influence of different carbon and nitrogen combinations in the medium for T. harzianum production. T. harzianum was cultivated in Erlenmeyer flasks and the effect of cultivation broth against selected maize pathogens was tested using well diffusion method. The results of this study showed that the combination of different carbon and nitrogen sources in the T. harzianum cultivation medium statistically significantly affects the production of Trichoderma cultivation broth effective on two tested phytopathogens. Dextrose as a carbon source and soybean flour as a nitrogen source proved to be the best combination in the medium for production of T. harzianum cultivation broth effective on A. flavus and F. graminearum. Maximal inhibition zone diameters of 31 mm and 56.33 mm were registered in those medium formulations for A. flavus and F. graminearum, respectively. These researches represent an important step for further research in which a medium of low market value would be selected. This would reduce the price of the production process but also the final product.

Discovery of a Potent and Dual-Selective Bisubstrate Inhibitor for Protein Arginine Methyltransferase 4/5

Protein arginine methyltransferases (PRMTs) have been implicated in the progression of many diseases. Understanding substrate recognition and specificity of individual PRMT would facilitate the discovery of selective inhibitors towards future drug discovery. Herein, we reported the design and synthesis of bisubstrate analogues for PRMTs that incorporate a S-adenosylmethionine (SAM) analogue moiety and a tripeptide through an alkyl substituted guanidino group. Compound AH237 is a potent and selective inhibitor for PRMT4 and PRMT5 with a half-maximal inhibition concentration (IC50) of 2.8 nM and <1.5 nM, respectively. Computational studies provided a plausible explanation for the high potency and selectivity of AH237 for PRMT4/5 over other 40 methyltransferases. This proof-of-principle study outlines an applicable strategy to develop potent and selective bisubstrate inhibitors for PRMTs, providing valuable probes for future structural studies.

A Gap Prepulse with a Principal Stimulus Yields a Combined Auditory Late Response

Background and Objectives: The gap prepulse inhibition of the acoustic startle response has been used to screen tinnitus in an animal model. Here, we examined changes in the auditory late response under various conditions of gap prepulse inhibition.Subjects and Methods: We recruited 19 healthy adults (5 males, 14 females) and their auditory late responses were recorded after various stimuli with or without gap prepulsing. The N1 and P2 responses were selected for analysis. The gap prepulse inhibition was estimated to determine the optimal auditory late response in the gap prepulse paradigm.Results: We found that the gap per se generated a response that was very similar to the response elicited by sound stimuli. This critically affected the gap associated with the maximal inhibition of the stimulus response. Among the various gap-stimulus intervals (GSIs) between the gap and principal stimulus, the GSI of 150 ms maximally inhibited the response. However, after zero padding was used to minimize artifacts after a P2 response to a gap stimulus, the differences among the GSIs disappeared.Conclusions: Overall, the data suggest that both the prepulse inhibition and the gap per se should be considered when using the gap prepulse paradigm to assess tinnitus in humans.

89 Evaluating the effect of Ca-gluconate and Ca-butyrate on SCFA absorption and permeability of the gastrointestinal tract

The objective was to evaluate short-chain fatty acid (SCFA) absorption and permeability of the gastrointestinal tract (GIT) of heifers infused either with Ca-gluconate or Ca-butyrate. Thirty-two ruminally cannulated beef heifers were fed a common diet (forage-to-concentrate ratio of 50:50) for 28 d and once daily infused with water (ruminal infusion; control), Ca-gluconate embedded in a fat matrix (ruminal infusion; 0.192% BW), unprotected Ca-gluconate (abomasal infusion; 0.077% of BW), and unprotected Ca-butyrate (abomasal infusion; 0.029% BW). Treatments were designed to provide the same amount of butyrate to the small intestine. DMI was restricted to 95% of voluntary DMI on d 8 and was recorded until heifers were slaughtered on d 28. Rumen, jejunum, and colon tissues were collected to determine the rate and pathway of SCFA transport, and for measurement of permeability.14C-acetate and 3H-butyrate absorption across the ruminal and colonic epithelium was measured with no inhibition and under maximal inhibition. Permeability was assessed for the rumen, jejunum and colon using mucosal-to-serosal flux of 14C-mannitol. Initial and final BW were not different (P &gt; 0.60) averaging 388 ± 5.5 kg and 409 ± 6.6 kg, respectively. DMI was not affected by treatment averaging 7.7 kg/d (P = 0.77). Treatment did not affect the flux of acetate or butyrate across the ruminal or colonic epithelium (P &gt; 0.33), but flux rates of SCFA were numerically greater across the ruminal epithelium. Moreover, approximately 50% of the ruminal acetate was transported via bicarbonate-dependent mechanisms. Mannitol flux was not affected by treatment (P &gt; 0.29) and was numerically lower in the rumen and colon than in the jejunum, supporting previous research evaluating permeability across the GIT. According to this experiment, provision of Ca-butyrate or Ca-gluconate in an attempt to increase intestinal butyrate supply does not impact SCFA absorption or permeability of the rumen, jejunum, or colon.

Solid-state fermentation as an efficient strategy for the biotransformation of lentils: enhancing their antioxidant and antidiabetic potentials

Background Fermentation is a classic industrial process that can be applied as an efficient strategy to increase the release of bioactive compounds with antioxidant and antidiabetic activities. Methods This work reported the effects of solid-state fermentation (SSF) performed using strains of Aspergillus oryzae and Aspergillus niger on the antioxidant (DPPH, ABTS and FRAP) and in vitro antidiabetic (inhibition of α-amylase and α-glucosidase activities) potential of lentils. Results The results showed that the profiles of the biological activities of the extracts obtained from the fermented samples varied greatly with respect to both the microorganism involved and the fermentation time. The extracts obtained from the fermented lentils by A. oryzae after 72 h and by A. niger after 48 h using the FRAP assay showed the most remarkable changes in the antioxidant activity, increasing by 107 and 81%, respectively, compared to the nonfermented lentils. The lentil extracts produced by fermentation with A. niger after 48 h were able to inhibit the α-glucosidase activity by up to 90%, while a maximal inhibition of amylase (~ 75%) was achieved by the lentil extract obtained after 24 h of fermentation with A. oryzae. The content of the total phenolic compounds (TPCs) and the identification of them in lentil extracts correlated well with the improvement of the biological activities. Conclusion These results suggested that SSF was feasible to obtain extracts of fermented lentils with improved antioxidant and antidiabetic properties. Additionally, these results indicated that the proper choice of microorganism is crucial to direct the process for the production of compounds with specific biological activities.

LH2 complexes (B800-850 AND B800-830) are assembled in the cells of sulfur bacterium Thiorhodospira sibirica, strain Kir 3, without any carotenoids

It has been studied the result of assembling the light-harvesting complexes in the cells of purple sulfuric bacterium Thiorhodospira (T.) sibirica, strain Kir‑3, while suppressing biosynthesis of carotenoids by diphenylamine (DPA). LH2 complexes (B800-850 and B800-830) with different carotenoids’ composition were isolated from the cells obtained. Maximal inhibition of carotenoid biosynthesis (~90% of the control) was achieved at the inhibitor concentration of 53,25 mM (9 mg/l). It has been established that changes in qualitative and quantitative composition of carotenoids do not affect the assembling of B800-830 and B800-850 complexes. It is assumed that in the population of DPA-LH2 complexes from T. sibirica, strain Kir‑3, both carotenoidless complexes and the complexes, containing one or two carotenoid molecules, can be assembled. These results support a hypothesis that carotenoids are not required for assembling B800-850 and B800-830 complexes.