The Development and Survival of Thymic Epithelial Cells Require TSC1-Dependent Negative Regulation of mTORC1 Activity.

Thymic epithelial cells (TECs) are critical for the development and generation of functionally competent T cells. Until now, the mechanism that regulates the survival of TECs is poorly understood. In the current study, we found that Tsc1 controls the homeostasis of medullary TECs (mTECs) by inhibiting lysosomal-mediated apoptosis pathway in mice. TEC-specific deletion of Tsc1 predominately decreased the cell number of mTECs and, to a lesser content, affected the development cortical TECs. The defect of mTECs caused by Tsc1 deficiency in mice impaired thymocyte development and peripheral T cell homeostasis. Mechanistically, Tsc1 deficiency did not affect the cell proliferation of mTECs but increased the apoptosis of mTECs significantly. RNA-sequencing analysis showed that pathways involved in lysosomal biogenesis, cell metabolism, and apoptosis were remarkably elevated in Tsc1-deficient mTECs compared with their wild-type counterparts. Tsc1-deficient mTECs exhibited overproduction of reactive oxygen species and malfunction of lysosome, with lysosome membrane permeabilization and the release of cathepsin B and cathepsin L to the cytosol, which then lead to Bid cleaved into active truncated Bid and subsequently intrinsic apoptosis. Finally, we showed that the impaired development of mTECs could be partially reversed by decreasing mTORC1 activity via haploinsufficiency of Raptor Thus, Tsc1 is essential for the homeostasis of mTECs by inhibiting lysosomal-mediated apoptosis through mTORC1-dependent pathways.

Effect of Low-Temperature Phosphine Fumigation on the Survival of Bactrocera correcta (Diptera: Tephritidae).

This laboratory-based study examined the effects of low-temperature phosphine fumigation on the survival of the eggs and larvae of the guava fruit fly, Bactrocera correcta (Bezzi). Individual flies at different developmental stages, from 6-h-old eggs to third instars, were exposed to 0.92 mg/liter phosphine for 1-7 d at 5°C. We found that 12-h-old eggs and third instars were the most tolerant to phosphine. Increasing phosphine concentrations from 0.46 to 4.56 mg/liter increased mortality in these two stages. However, increased exposure times were required to achieve equal mortality rates in 12-h-old eggs and third instars when phosphine concentrations were ≥4.56 and ≥3.65 mg/liter, respectively. C(n)t = k expression was obtained at 50, 90, and 99% mortality levels, and the toxicity index (n) ranged from 0.43 to 0.77 for the two stages. The synergistic effects of a controlled atmosphere (CA) with elevated CO(2) levels were also investigated, and we found that a CO(2) concentration between 10% and 15% under CA conditions was optimal for low-temperature phosphine fumigation.

Regulation of the kiss2 promoter in yellowtail clownfish (Amphiprion clarkii) by cortisol via GRE-dependent GR pathway.

Kisspeptin plays a vital role in mediating the stress-induced reproductive regulation. Cortisol, known as a stress-related hormone, is involved in gonadal development and sexual differentiation by binding with glucocorticoid receptor (GR) to regulate the expression of kiss gene. In the present study, cortisol treatment in yellowtail clownfish (Amphiprion clarkii) showed that the expression of kiss (kiss1 and kiss2) and gr (gr1 and gr2) genes were increased significantly. We demonstrated that the yellowtail clownfish Kiss neurons co-express the glucocorticoid receptors in the telencephalon, mesencephalon, cerebellum, and hypothalamus. We further cloned the promoter of kiss2 gene in yellowtail clownfish and identified the presence of putative binding sites for glucocorticoid receptors, estrogen receptors, androgen receptors, progesterone receptors, AP1, and C/EBP. Applying transient transfection in HEK293T cells of the yellowtail clownfish kiss2 promoter, cortisol (dexamethasone) treatment was shown to enhance the promoter activities of the yellowtail clownfish kiss2 gene in the presence of GRs. Deletion analysis of kiss2 promoter indicated that cortisol-induced promoter activities were located between position -660 and -433 with GR1, and -912 and -775 with GR2, respectively. Finally, point mutation studies on the kiss2 promoter showed that cortisol-stimulated promoter activity was mediated by one GRE site located at position -573 in the presence of GR1 and by each GRE site located at position -883, -860, -851, and -843 in the presence of GR2. Results of the present study provide novel evidence that cortisol could regulate the transcription of kiss2 gene in the yellowtail clownfish via GRE-dependent GR pathway.

Sexually dimorphic distribution of kiss1 and kiss2 in the brain of yellowtail clownfish, Amphiprion clarkii.

Kisspeptin system was shown to be a key factor in mediating social stress and reproduction. Yellowtail clownfish, Amphiprion clarkii, is a hermaphrodite fish, whose sex determination and gonadal development are affected by the social status of individuals. The yellowtail clownfish is a fantastic animal model to explore sex determination, but the social status and precise distribution of kiss mRNAs in the brain of this species are unknown. Hererin, a novel in situ hybridization technique, RNAscope, was used to investigate the distribution of kiss1 and kiss2 expressions in the brain of yellowtail clownfish. The coronal planes of brain showed that the kiss1 signal was mainly present in dorsal habenular nucleus (NHd) and kiss2 mRNA was widely expressed in telencephalon, midbrain, and hypothalamus, especially in dorsal part of the nucleus of the lateral recess (NRLd). Additionally, kiss1 and kiss2 signals have sexually dimorphic distribution. The kiss1 mRNA was distributed in NHd, the telencephalon, and lateral part of the diffuse nucleus of the inferior lobe (NDLIl) of females but in NHd and NDLIl of males. kiss2 signals were stronger in females than that in males. The distribution of kiss1 and kiss2 neurons in NHd of habenula and NRLd of hypothalamus may suggest that kiss genes associate environmental signaling and reproductive function in yellowtail clownfish.

Combination of Modified Atmosphere and Irradiation for the Phytosanitary Disinfestation of Trogoderma granarium Everts (Coleoptera: Dermestidae).

The khapra beetle, Trogoderma granarium Everts, is defined as one of the most important quarantine pests globally, and fumigation with methyl bromide, an ozone-depleting substance, is a common phytosanitary measure currently used. The modified atmosphere (MA), irradiation, and their combination treatments of T. granarium larvae and adults were performed at room temperature (24-26 ℃) to develop an ecofriendly phytosanitary disinfestation measure and to shorten the exposure time and overcome treatment disadvantages of irradiation. Late-stage larvae are determined as the most tolerant stage resulted in large LT99.9968 values of 32.6 (29.2-37.5) and 38.0 (35.1-41.7) days treated under 1% and 2% O2 (with N2 balance) atmosphere, respectively. Ionizing radiation was used to enhance the effect of MA and the mortality was highly significantly affected by all the interaction effects, indicating that the synergistic effects present in all the combined treatments. The synergistic ratios, which is defined as the estimated lethal time for MA treatment (LD90, LD99, and LD99.9968), divided by that of combined treatment, were between 1.47 and 2.47. In the confirmatory tests, no individuals recovered from a sum of 111,366 late-stage larvae treated under 1% O2 atmosphere for 14- or 15-d after 200 Gy irradiation, which resulted in validating the probit estimations and achieving an efficacy of 99.9973% mortality at 95% confidence level. Therefore, these treatment schedules are recommended to disinfest T. granarium infecting commodities for phytosanitary purposes under the warehouse, MA packaging, or in combination with international transportation by train or sea container.