[Killing effect of anti-MSLN-iCAR-NK cells derived from induced pluripotent stem cells on ovarian epithelial cancer cells].

Objective: To investigate the cytotoxic effects of induced pluripotent stem (iPS) cells of anti-mesothelin (MSLN)-chimeric antigen receptor natural killer (CAR-NK) cells (anti-MSLN-iCAR-NK cells) on ovarian epithelial cancer cells. Methods: Twenty cases of ovarian cancer patients who underwent surgical treatment at Henan Provincial People's Hospital from September 2020 to September 2021 were collected, and 20 cases of normal ovarian tissues resected during the same period due to other benign diseases were also collected. (1) Immunohistochemistry and immunofluorescence were used to verify the expression of MSLN protein in ovarian cancer tissues. (2) Fresh ovarian cancer tissues were extracted and cultured to obtain primary ovarian cancer cells. Recombinant lentiviral vectors targeting anti-MSLN-CAR-CD244 were constructed and co-cultured with iPS cells to obtain anti-MSLN-iCAR cells. These cells were differentiated into anti-MSLN-iCAR-NK cells using cytokine-induced differentiation method. The cell experiments were divided into three groups: anti-MSLN-iCAR-NK cell group, natural killer (NK) cell group, and control group. (3) Flow cytometry and live cell staining experiment were used to detect the apoptosis of ovarian cancer cells in the three groups. (4) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B (GZMB), perforin 1 (PRF1), interleukin (IL)-6, and IL-10 in the three groups of ovarian cancer cells. Results: (1) Immunohistochemistry analysis showed that a positive expression rate of MSLN protein in ovarian cancer tissues of 65% (13/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=4.912, P=0.027). Immunofluorescence analysis revealed that the positive expression rate of MSLN protein in ovarian cancer tissues was 70% (14/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=6.400, P=0.011). (2) Flow cytometry analysis showed that the apoptotic rate of ovarian cancer cells in the anti-MSLN-iCAR-NK cell group was (29.27±0.85)%, while in the NK cell group and control group were (8.44±0.34)% and (6.83±0.26)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.01). Live cell staining experiment showed that the ratio of dead cells to live cells in the anti-MSLN-iCAR-NK cell group was (36.3±8.3)%, while in the NK cell group and control group were (5.4±1.4)% and (2.0±1.3)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.001). (3) ELISA analysis revealed that the expression levels of IFN-γ, TNF-α, GZMB, PRF1, IL-6, and IL-10 in ovarian cancer cells of the anti-MSLN-iCAR-NK cell group were significantly higher than those in the NK cell group and the control group (all P<0.05). Conclusion: The anti-MSLN-iCAR-NK cells exhibit a strong killing ability against ovarian cancer cells, indicating their potential as a novel immunotherapy approach for ovarian cancer.

[Associations between maternal exposure to chemical fertilizers during pregnancy and the risk of offspring's low birth weights].

Objective: To explore the association between the consumption of chemical fertilizers and the risk of low birth weight (LBW), to provide references for prevention programs on LBW and to improve the birth outcomes. Methods: Stratified multivariate logistic regression method was used in this study involving 153 preterm LBW infants, 179 term LBW infants and 204 normal control infants that were randomly selected from the birth monitoring data between October 2007 and September 2012 in Pingding county, Shanxi province. Associations between the risk of LBW and maternal exposure to chemical fertilizers during pregnancy were identified. A normal control group was set up to compare results between preterm and term LBW groups. Results: Totally, 18 749 infants were born between 2007 and 2012, with the total incidence rates of LBW as 48.5‰, preterm LBW as 19.4‰, and term LBW as 29.1‰. Concerning the case control study on preterm LBW, after adjustment for confounding factors, the risk of preterm LBW appeared 2.51 (95%CI: 1.05-5.99) times higher in villages with annual consumption of chemical fertilizer ≥100 tons than those villages that using chemical fertilizer less than 50 tons. No significant statistical associations were found between the amounts of household chemical fertilizer consumption and the risks of preterm LBW. Regarding the case control study on term LBW, after adjustment for confounding factors, in villages with ≥100 tons annual consumption of chemical fertilizers, the risk of term LBW was 4.03 (95%CI: 1.63-9.92) times of the risk in villages where the annal use of chemical fertilizers was less than 50 tons. There was no significant association between household consumption of chemical fertilizers and the risk of term LBW. Conclusions: Maternal exposure to chemical fertilizers during pregnancy was associated with the risk of LBW. Our findings suggested that the amount of chemical fertilizer consumption in rural areas seemed also associated with the risks of other adverse pregnancy outcomes. Women should avoid the chance of exposure to chemical fertilizers during pregnancy and the consumption of chemical fertilizers should be carefully managed.

Olfactory discrimination conditioning in the moth Spodoptera littoralis.

We used a proboscis extension reflex (PER) to study the olfactory discrimination capability in the moth Spodoptera littoralis. Already after a single experience, moths were capable to discriminate a rewarded from an unrewarded odor. In the first experiment, when rewarded and unrewarded odors were substituted for each other, moths were able to undergo reversal conditioning already after two experiences. Both shorter and longer inter-trial intervals (ITIs) supported high degrees of learning. In a second experiment, moths could solve both feature-positive and -negative discrimination tasks. Two hypotheses for the way in which these associations exert their discrimination performance are considered. The moth's olfactory physiology has been extensively studied. This animal thus provides a powerful system in which to study the neurobiology of olfactory discrimination and odor recognition.

Analysis of nocifensive behavior induced in rats by CO2 laser pulse stimulation.

To characterize nocifensive behavior, a laser beam was applied to the hind footpad of nonanesthetized and unrestrained rats and the reaction pattern was analyzed. Fifty-four rats were divided into nine groups of six animals, and each group was given one of nine combinations of laser stimuli: intensity of 4, 8 or 12 W and duration of 10, 30, or 50 ms. A single pulse was applied to a 0.13 cm2 area of right or left footpad and the trial was repeated 20 times with 3 min between trials. The behavior was videotaped and reviewed for a period of 2 min following each stimulation. It seemed to consist of eight discrete responses, and each response was scored for whether it occurred and for its summed duration per trial. The component responses and the behavior as a whole were characterized by their sensitivity in terms of the level of energy required to attain 50% of the maximum response, and their linear or quadratic trends with increasing stimulus energy. The most sensitive index of pain stimulation was the composite score, followed by foot jumping, foot elevation, body movements, licking, and then foot movements. As stimulus energy increased, rats exhibited a greater number of different responses and a greater frequency of each component response. The results suggest that a pool of hierarchically organized responses in the nocifensive motor system are recruited partially or wholly by nociceptive stimuli of varying intensity.

Additivity of taste-specific effects of sucrose and quinine: microstructural analysis of ingestive behavior in rats.

Effects of sweet and bitter tastes on ingestion were studied by timing licking responses. Twelve water-deprived rats were given 15-min access to sucrose (S) solutions (0.00%, 1.25%, 2.50%, and 5.00%) with and without quinine (0.01%) and to quinine (Q) solutions (0.00%, 0.0025%, 0.005%, and 0.01%) with and without sucrose (5.00%). Volume ingested and number of licks increased with S and decreased with Q. In response to S, the number of bursts increased, and interlick intervals lengthened. In response to Q, licks to ingest 1 ml of solution, burst number, and percentage of slow licks increased, and burst size decreased. When Q and S were mixed in the same solution, the pattern of ingestive responses manifested attributes of both tastes. Results suggest 2 separate, parallel systems that operate simultaneously to govern rats' licking behavior. One system expresses the effect of S on the pattern of ingestion and the other expresses the effects of Q.