TGF-ß1-induced collagen promotes chicken ovarian follicle development via an intercellular cooperative pattern.

Follicle development is a complex process under strict regulation of diverse hormones and cytokines including transforming growth factor ß (TGF-ß) superfamily members. TGF-ß is pivotal for the regulation of ovarian functions under physiological and pathological conditions. In this study, effect of TGF-ß1 on chicken follicle development was examined through investigating the accumulation and action of collagen, an indispensable member of the extracellular matrix (ECM) involved in this process. The granulosa cells (GCs) and theca cells (TCs) were separated from growing follicles of the laying chicken for treatment of TGF-ß1 and analysis of expression of ECM components and key proteins in intracellular signaling pathways. Results showed that collagen was mainly distributed in the follicular theca layer and was produced with the formation of the granulosa layer during ovarian development. Collagen accumulation increased with follicle growth and treatment of GCs with TGF-ß1 elicited an increased expression of collagen. After production from GCs, collagen was transferred to the neighboring TCs to promote cell proliferation and inhibit apoptosis. Treatment of collagen remarkably increased expression of p-ERK, mitogen-activated protein kinase (MAPK), and p-MAPK, but treatment with hydroxylase inhibitor (to break collagen structure) reversed these alterations. In conclusion, during follicle growth collagen was secreted by GCs under TGF-ß1 stimulation and was subsequently collaboratively transferred to neighboring TCs to increase cell proliferation and thus to promote follicle development via an intercellular cooperative pattern during development of chicken growing follicles.

Instruction-Level Power Side-Channel Leakage Evaluation of Soft-Core CPUs on Shared FPGAs.

Side-channel disassembly attacks recover CPU instructions from power or electromagnetic side-channel traces measured during code execution. These attacks typically rely on physical access, proximity to the victim device, and high sampling rate measuring instruments. In this work, however, we analyze the CPU instruction-level power side-channel leakage in an environment that lacks physical access or expensive measuring equipment. We show that instruction leakage is present even in a multitenant FPGA scenario, where the victim uses a soft-core CPU, and the adversary deploys on-chip voltage-fluctuation sensors. Unlike previous remote power side-channel attacks, which either require a considerable number of victim traces or attack large victim circuits such as machine learning accelerators, we take an evaluator's point of view and provide an analysis of the instruction-level power side-channel leakage of a small open-source RISC-V soft processor core. To investigate whether the power side-channel traces leak secrets, we profile the victim device and implement various instruction opcode classifiers based on both classical machine learning algorithms used in disassembly attacks, and novel, deep learning approaches. We explore how parameters such as placement, trace averaging, profiling templates, and different FPGA families (including a cloud-scale FPGA) impact the classification accuracy. Despite the limited leakage of the soft-core CPU victim and a reduced accuracy and sampling rate of on-chip sensors, we show that in a worst-case scenario for the evaluator, i.e., an attacker breaching physical separation, we can identify the opcode of executed instructions with an average accuracy as high as 86.46%. Our analysis shows that determining the executed instruction type is not a classification bottleneck, while leakages between instructions of the same type can be challenging for deep learning models to distinguish. We also show that the instruction-level leakage is significantly reduced in a cloud-scale FPGA scenario with higher soft-core CPU frequencies. Nevertheless, our results show that even small circuits, such as soft-core CPUs, leak potentially exploitable information through on-chip power side channels, and users should deploy mitigation techniques against disassembly attacks to protect their proprietary code and data.

Correlation Analyses on Physical and Mechanical Parameters of Concrete in Marine Environments.

This work explored the correlation among the physical and mechanical parameters of concrete in marine environments during corrosion. Concrete materials with varied water-cement ratios (w/c) were soaked in four kinds of simulated seawater, including a compound solution of sodium chloride (NaCl) and magnesium sulfate (MgSO4), a solution of MgSO4, a solution of sodium sulfate (Na2SO4), and clear water. The macroscopic physical and mechanical parameters, such as compressive strength, tensile strength, mass change and ultrasonic wave velocity, were measured. The damage mechanism of corrosion products and microstructures was analyzed using microscopic approaches including scanning electron microscopy (SEM), X-ray diffraction (XRD), etc. Linear correlation was carried out on the abovementioned physical parameters, and the results revealed that, during corrosion, the correlation between compressive and tensile strength and mass (excluding Na2SO4 solution) is positive (mostly highly correlated), while that with wave velocity is poor (mostly moderately correlated).

Naringin prevents follicular atresia by inhibiting oxidative stress in the aging chicken.

Oxidative stress is an essential inducement in follicle atresia and ovarian aging, resulting in decline in female fecundity. As a natural and effective antioxidant, naringin was investigated to relieve chicken follicle atresia and ovarian aging. First, the cultured small white follicles (SWFs) from D280 hens were pretreated with 0.5 mM naringin for 24 h and then treated with H2O2 for 72 h to establish the oxidative stress model to evaluate the putative attenuating effects of naringin on follicle atresia. Meanwhile, SWFs of D580 hens were treated with naringin for 72 h to examine the attenuating effect on the physiological aging of SWFs. Finally, each hen was fed with naringin at a dose of 50 mg/kg every day to explore the effect of naringin on follicular development and laying performance in D580 hens. Results showed that naringin could rescue the antioxidant capacity decline by increasing the antioxidant-related indexes and expression of antioxidation-associated genes. It could also maintain the homeostasis of SWFs in both the H2O2-induced group and natural physiological aging group. In addition, naringin increased estrogen levels, capacity of antioxidants, and the laying performance in aged laying chickens. The thickness and strength of the eggshell were increased in the naringin-treated group as well. In conclusion, this study showed that naringin is capable of relieving SWFs atresia that was induced by oxidative stress and maintaining the laying performance of aging low-yielding hens by reducing oxidative stress.

Enhancing effect of FSH on follicular development through yolk formation and deposition in the low-yield laying chickens.

Healthy and efficient development of ovarian follicles largely determines poultry laying performance. In low-yield laying chickens, retarded follicle progression resulted in decreased prehierarchical follicles. In this study the extenuating effect of follicle-stimulating hormone (FSH) on delayed follicular development was investigated in the low-yield chickens. Results showed that FSH administration in vivo accelerated development of prehierarchical follicles, with increased expression of steroidogenic enzymes and follicular angiogenesis through elevating plasma levels of 17ß-estradiol, progesterone, luteinizing hormone and the expression of vascular endothelial growth factor and its receptor as well as angiopoietins. Furthermore, treatment with FSH raised expression of lipid uptake and adipogenesis-related proteins and decreased tight junctions between granulosa cells. Meanwhile, the results of the in vivo studies were confirmed by the in vitro studies as FSH promoted development of the cultured prehierarchical follicles with increased angiogenesis, cell proliferation, steroid hormones synthesis and yolk deposition. These results indicated FSH enhanced follicular development in the low-yield laying chickens involving increased follicular angiogenesis.

Improvement of eggshell quality by dietary N-carbamylglutamate supplementation in laying chickens.

Egg quality defects seriously reduce the quality grade and increase egg breakage in egg marketing activities. In this study, the effect of N-carbamylglutamate (NCG) on eggshell quality was investigated by evaluating calcium absorption and calcification in laying hens. A total of 30 newly hatched female Hy-Line chicks were randomly assigned to the control group (basal diet) and treatment group (basal diet supplemented with 1% NCG). At 25 wk, eggs from each group were obtained to assess egg quality parameters. Blood samples were collected for analysis of mineral, hormone, and amino acids levels at 16 h after laying egg. Uterine tissues were removed and fixed in 4% neutral paraformaldehyde solution or kept in liquid nitrogen for mineral determination, quantitative PCR, and Western blot. Results showed that the egg quality (eggshell thickness, strength and percentage, egg specific gravity, and eggshell effective thickness) was significantly increased while effective thickness of mastoid layer, width of mastoid gap, and mammillary knobs were significantly decreased by dietary NCG supplementation (P < 0.05). The levels of minerals (Ca, P, Fe, Mg, Na, K) in eggshell, plasma, and uterus were remarkably elevated (P < 0.05). Meanwhile, the concentrations of calcium metabolism-related hormones (17ß-estradiol, parathyroid hormone, and calcitonin) were increased in the NCG group (P < 0.05). Moreover, expression of calbindin 1, carbonic anhydrase 2, ovalbumin, ovotransferrin, ovocleidin-17, ovocleidin-116, and clusterin mRNAs, as well as calbindin 1 and ATP2A1 proteins in uterus, duodenum, and kidney, was all upregulated in hens fed with NCG (P < 0.05). In addition, the number of blood vessels in the uterus, height of uterine mucosal folds, villus length in endometrium, and areas of uterine mucosal folds were significantly increased in the NCG group (P < 0.05). In conclusion, dietary 1% NCG supplementation during 0 to 25 wk can improve eggshell quality through changes in endometrial morphology, expression of calcium metabolism-related genes, and secretion of related hormones to promote eggshell formation in the laying hens.

The Attenuating Effect of the Intraovarian Bone Morphogenetic Protein 4 on Age-Related Endoplasmic Reticulum Stress in Chicken Follicular Cells.

In the poultry, only less than 5% primordial follicles in the ovary can develop into the prehierarchical follicles (PHFs) leading to progressive development, ovulation, and egg formation. This low rate of recruitment indicates a huge potential for improvement of the laying performance. A great reduction in egg production is caused by aging with extensive follicular atresia. In this study, age-related changes in the laying performance and ovarian status were compared between the peak-lay (D280) and aged (D580) chickens. Subsequently, a cross coculture of PHFs and granulosa cells (GCs) from D280 or D580 hens was adopted to reveal the mechanism of declined follicle development. Results showed that persistent endoplasmic reticulum (ER) stress in GCs of the aged hens was accompanied with intensified apoptosis. Bone morphogenetic protein 4 (BMP4) secreted by GCs of PHFs in D280 hens was capable of relieving ER stress and improving follicular dominance for selection in D580 hens. During this action, BMP4 reduced free calreticulin (CALR, an ER marker) content and attenuated cell apoptosis in PHFs of D580 hens via the PERK-CHOP-BCL2/caspase3 or CALR-Ca2+-BCL2-caspase12 pathway. Furthermore, BMP4 prevented follicular atresia by promoting production of steroid hormones to improve survival of GCs in PHFs from the aged hens. In conclusion, intensified ER stress and apoptosis occurred in GCs of PHFs in aged chickens, while BMP4 secreted by GCs was capable of improving follicular viability by alleviating ER stress to promote follicular development.

Down-regulation of PCK2 inhibits the invasion and metastasis of laryngeal carcinoma cells.

Laryngeal carcinoma is one of the common malignancies of head and neck. However, the pathogenesis of laryngeal cancer has been not completely clear. To identify the effects of hypoxia on the invasion, metastasis, and metabolism of laryngeal carcinoma, iTRAQ-labeling-with-LC-MS/MS analysis was performed to identify differentially expressed proteins of the SCC10A cells under hypoxia and normoxia, while metabolites were examined by metabolic profiling. 155 proteins and 180 metabolites were identified and the PCK2 protein was selected for validation by Western Blotting. Immunohistochemistry (IHC) was performed to analyze the expression of PCK2 in formalin-fixed paraffin-embedded (FFPE) tissue sections, including laryngeal squamous cell carcinoma tissues from various stages. Collectively, we report that down-regulation of PCK2 inhibits the invasion, migration, and proliferation of laryngeal cancer under hypoxia and down-regulation of PCK2 may be used as a new strategy for laryngeal cancer therapy.

Metformin Prevents Follicular Atresia in Aging Laying Chickens through Activation of PI3K/AKT and Calcium Signaling Pathways.

Increased follicular atresia occurs with aging and results in reduced fecundity in laying chickens. Therefore, relieving follicular atresia of aging poultry is a crucial measure to maintain sustained high laying performance. As an antiaging agent, metformin was reported to play important roles in preventing aging in diverse animals. In this study, the physiological state of the prehierarchical follicles in the peak-laying hens (D280) and aged hens (D580) was compared, followed with exploration for the possible capacity of metformin in delaying atresia of the prehierarchical follicles in the aged D580 hens. Results showed that the capacity of yolk deposition within follicles declined with aging, and the point of endoplasmic reticulum- (ER-) mitochondrion contact decreased in the ultrastructure of the follicular cells. Meanwhile, the expression of apoptosis signaling genes was increased in the atretic small white follicles. Subsequently, the H2O2-induced follicular atresia model was established to evaluate the enhancing capacity of metformin on yolk deposition and inhibition of apoptosis in the atretic small white follicles. Metformin inhibited apoptosis through regulating cooperation of the mitochondrion-associated ER membranes and the insulin (PI3K/AKT) signaling pathway. Furthermore, metformin regulated calcium ion homeostasis to relieve ER-stress and inhibited release of mitochondrion apoptosis factors (BAD and caspase). Additionally, metformin activated PI3K/AKT that suppressed activation of BAD (downstream of the insulin signaling pathway) in the atretic follicles. Further, serum estrogen level and liver estrogen receptor-α expression were increased after dietary metformin supplementation in D580 hens. These results indicated that administration of dietary metformin activated the PI3K/AKT and calcium signaling pathway and enhanced yolk deposition to prevent chicken follicular atresia.

Fractal Cracking Patterns in Concretes Exposed to Sulfate Attack.

Sulfate attack tests were performed on concrete samples with three water-to-cement ratios, and micro-crack growth patterns on concrete surfaces were recorded. The expansive stress and crack nucleation caused by delayed ettringite formation (DEF) were studied using X-ray diffraction and scanning electron microscopy. By means of a digital image processing technology, fractal dimensions of surface cracking patterns were determined, which monotonously increase during corrosion. Moreover, it is shown that the change of fractal dimensions is directly proportional to accumulation of DEF, and therefore, a simple theoretical model could be proposed to describe the micro-crack evolution in concretes under sulfate attack.

Basic fibroblast growth factor promotes prehierarchical follicle growth and yolk deposition in the chicken.

Basic fibroblast growth factor (bFGF) plays a pivotal role in prompting ovarian follicular development and angiogenesis as well as inhibiting atresia. In the chicken, high laying performance depends largely on efficient healthy development of ovarian follicles. Moreover, rapid growth of oocytes resulted from abundant yolk deposition via blood circulation and intra-ovarian interactions among somatic and germ cells. The major components of yolk mass consist of very low density lipoprotein (VLDL) and vitellogenin that are taken up by maturing oocytes via VLDL receptor (VLDLR)-mediated endocytosis from blood capillaries in the theca layer and gaps between granulosa cells. Here we used immunofluorescence, BrdU, TUNEL, Western bolt and RT-qPCR methods to investigate effects of bFGF on growth and yolk deposition of chicken prehierarchical follicles. Results showed that VLDLR was mainly expressed in the granulosa cells of the prehierarchical and preovulatory follicles, and its expression declined with follicle growth. Moreover, bFGF caused a dose-dependent promoting effect on growth of small white follicles and this effect was inhibited by SU5402 (an FGFR1 antagonist). Proliferation of follicular theca externa cells was accelerated by bFGF via FGFR1-AKT signaling, coupled with augmented angiogenesis and up-regulated p-ERK expression in granulosa cells. After combined inhibition of FGFR1 and PPARγ, we found that PPARγ could also suppress VLDLR expression in granulosa cells. These results indicate that bFGF facilitated growth and yolk deposition in chicken prehierarchical follicles through promoting proliferation and angiogenesis in theca layers, and also through down-regulating VLDLR expression in granulosa cells.