Reverse transcription‐quantitative (RT‐q) PCR is the most feasible and useful technique for identifying and evaluating cancer biomarkers; however, the method requires suitable reference genes for gene expression analysis. The aim of the present study was to identify the most suitable refer‐ ence gene for the normalization of relative gene expression in human hepatocellular carcinoma (HCC) tissue and blood samples. First, 14 candidate reference genes were selected through a systematic literature search. The expression levels of these genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, PGK1, PPIA, RPLP0, RPL13A, SDHA, TBP, TFRC and YWHAZ) were evaluated using human multistage HCC tran‐ scriptome data (dataset GSE114564), which included normal liver (n=15), chronic hepatitis (n=20), liver cirrhosis (n=10), and early (n=18) and advanced HCC (n=45). From the 14 selected genes, five genes, whose expression levels were stable in all liver disease statuses (ACTB, GAPDH, HMBS, PPIA and RPLP0), were further assessed using RT‐qPCR in 40 tissues (20 paired healthy tissues and 20 tissues from patients with HCC) and 40 blood samples (20 healthy controls and 20 samples from patients with HCC). BestKeeper statistical algorithms were used to identify the most stable reference genes, of which HMBS was found to be the most stable in both HCC tissues and blood samples. Therefore, the results of the present study suggest HMBS as a promising reference gene for the normalization of relative RT‐qPCR techniques in HCC‐related studies.

The objective of this study is to compare two experimental approaches to characterize the ebullition pressure (or air-entry suction) of initially water-saturated organophilic clay blankets. The first is an indirect approach using the water-retention curve (WRC) and the second is a direct approach using ebullition experiments. The WRC along with the hydraulic conductivity of organophilic clay blankets in saturated and unsaturated conditions were measured using a flexible-wall permeameter with suction-saturation control. This device was also adapted to measure the ebullition pressure and the air permeability. The comparison of the experimental approaches was performed on organophilic clay blanket specimens in different initial conditions (unrinsed and rinsed to remove loose fines) under high and low effective confining stresses (20 and 5 kPa). The indirect estimates of air-entry suction from the WRC were similar to those obtained from the ebullition tests. This good agreement between the two approaches may add flexibility to the development of design specifications for capping systems. The hydraulic properties were found to be sensitive to rinsing and effective stress, with greater hydraulic conductivity and air permeability for the rinsed specimen due to the removal of fines, and greater air-entry suctions for specimens under higher effective stress.

We explored a new feed ingredient which can be replaced with conventional protein supplements such as fish meal and blood meal for young monogastric animals. Insect meal, especially larvae of black soldier fly (BSF, Hermetia illucens) can possibly be a high-quality protein source due to the fact that BSF larvae have high crude protein (36.1%) and crude fat (36.5%) concentration. In trial 1 for weaning pigs (initial BW: 6.75 ± 0.10 kg on average), 2 treatments were tested: control (0% BSF) vs. 2% BSF. Feeding BSF at 2% increased average daily gain (ADG; 414 vs. 334 g/d) and average daily feed intake (ADFI; 496 vs. 405 g/d) compared with control, respectively. In trial 2 for weaning pigs (initial BW: 6.32 ± 0.08 kg on average), we evaluated the possibility of replacing spray-dried plasma protein (SDPP) with BSF having 3 treatments: 4% SDPP + 0% BSF, 2% SDPP + 2% BSF, and 0% SDPP + 4% BSF. Although ADG decreased by feeding BSF at 2 or 4% compared with control (295 vs. 324 g/d), ADFI and feed conversion ratio (FCR) were not affected by feeding BSF regardless of dietary concentration. Despite the negative impact on ADG, feeding BSF at 2 or 4% resulted in formulation of cost-effective diets. In trial 3 with layer chicks (initial BW: 40.1 ± 0.16 g), 3 treatments were assessed having BSF at 0, 3, and 5%. Layer chicks fed BSF at 3 or 5% increased ADG compared to those without BSF (10.3 vs. 9.49 g/d). While dietary treatments did not influence ADFI, FCR was improved by feeding BSF at 3 or 5% compared with the diet at 0% BSF (1.88 vs. 1.99). Overall results on the series of 3 trials suggest that feeding BSF up to 5% to young monogastric animals can improve productive performance.

Extended lactation may reduce the number of days dry within the animal lifetime and metabolic stress. The effects of an annual lactation cycle vs a two-year extended lactation were evaluated in 30 multiparous Murciano-Granadina dairy goats milked once-a-day throughout lactation. Goats were assigned to treatments at wk 29 of lactation and mated (M, n = 14) or kept open (O, n = 16). Milk yield (weekly from wk 2) and milk composition (biweekly from wk 30) were recorded up to wk 72. Cisternal and alveolar milk were evaluated at wk 39 (wk 10 of pregnancy) and 55 (wk 5 of subsequent lactation) by using an oxytocin receptor blocking agent (Tractocile, Ferring S.A., Madrid, Spain). Average milk yield during the first 29 wk was 2.28 L/d. Pregnancy reduced (P < 0.05) milk yield at wk 39 (1.19 vs 1.51), 40 (1.02 vs 1.53), 41 (0.55 vs 1.40), and 42 (0.30 vs 1.41) of lactation for M and O goats, respectively. From wk 43 to 50, M goats were in dry-off, whereas, O goats yielded 1.43 L/d. After kidding (wk 51 to 72), M goats produced 40% more milk than O goats (2.50 vs 1.51 L/d; P < 0.001). Milk of M goats contained lower (P < 0.05) log SCC (5.99 vs 6.49) than O goats. No significant changes were detected for fat, protein or lactose contents. Cisternal milk at wk 39 was lower for M than O goats (638 vs 1560 ml; P < 0.01), whereas, alveolar milk did not differ (345 ml). In the following lactation (wk 55) cisternal milk in M goats tripled (2063 ml) and was higher (P < 0.01) than in O goats (1218 ml). Similarly, alveolar milk doubled in M goats (680 ml) and was higher (P < 0.01) than in O goats (355 ml). Fat content was higher (P < 0.05) for alveolar milk (6.18%) than cisternal milk (3.74%) except for M goats at wk 39. No differences in percentages of protein and lactose, or in log SCC were detected between cisternal and alveolar milk, although cisternal milk of M goats contained lower SCC than alveolar milk at wk 55 (5.84 vs 6.09; P < 0.05). In conclusion, differences in milk yield between groups were clear in the last third of pregnancy and at the peak of the following lactation. Throughout 72 wk, extended lactation slightly decreased milk yield (6.9%) in our conditions.

The effect of glycyrrhizin (GL), a Chinese herbal drug extracted from licolice roots, on murine lymphocytes for inducing apoptotic cell death was studied. Addition of GL (25-400 micrograms/ml) to cultured splenocytes and thymocytes from BALB/c mice definitely promoted DNA fragmentation. A single injection of GL (100 micrograms/mouse) into BALB/c mice did not cause any detectable DNA fragmentation or cell death of splenocytes and thymocytes. Cytofluorometric analysis of these cells, however, demonstrated a reduction in mitochondrial transmembrane potentials (delta psi m). Repeated injections of GL (100 micrograms/mouse/day) into mice for 7 days actually resulted in induction of low grade DNA fragmentation selectively in splenocytes. Cell population analysis of viable lymphocytes suggested that both CD4+ Th lymphocytes and CD8+ Tc lymphocytes may have been relatively more sensitive than B220+ B lymphocytes for the apoptotic cell death. We concluded from these results that GL acts as a rather selective inducer of mature T lymphocyte apoptosis with a reduction in delta psi m potentially preceding lymphocyte death.