Showing papers by "Fuller W. Bazer published in 2016"

Amino acids and mammary gland development: nutritional implications for milk production and neonatal growth

Abstract: Milk is synthesized by mammary epithelial cells of lactating mammals. The synthetic capacity of the mammary gland depends largely on the number and efficiency of functional mammary epithelial cells. Structural development of the mammary gland occurs during fetal growth, prepubertal and post-pubertal periods, pregnancy, and lactation under the control of various hormones (particularly estrogen, growth hormone, insulin-like growth factor-I, progesterone, placental lactogen, and prolactin) in a species- and stage-dependent manner. Milk is essential for the growth, development, and health of neonates. Amino acids (AA), present in both free and peptide-bound forms, are the most abundant organic nutrients in the milk of farm animals. Uptake of AA from the arterial blood of the lactating dam is the ultimate source of proteins (primarily β-casein and α-lactalbumin) and bioactive nitrogenous metabolites in milk. Results of recent studies indicate extensive catabolism of branched-chain AA (leucine, isoleucine and valine) and arginine to synthesize glutamate, glutamine, alanine, aspartate, asparagine, proline, and polyamines. The formation of polypeptides from AA is regulated not only by hormones (e.g., prolactin, insulin and glucocorticoids) and the rate of blood flow across the lactating mammary gland, but also by concentrations of AA, lipids, glucose, vitamins and minerals in the maternal plasma, as well as the activation of the mechanistic (mammalian) target rapamycin signaling by certain AA (e.g., arginine, branched-chain AA, and glutamine). Knowledge of AA utilization (including metabolism) by mammary epithelial cells will enhance our fundamental understanding of lactation biology and has important implications for improving the efficiency of livestock production worldwide.

mTORC1 signaling and IL‐17 expression: Defining pathways and possible therapeutic targets

Abstract: IL-17 mediates immune responses against extracellular pathogens, and it is associated with the development and pathogenesis of various autoimmune diseases. The expression of IL-17 is regulated by various intracellular signaling cascades. Recently, it has been shown that mechanistic target of rapamycin (mTOR) signaling, comprised mainly of mTORC1 signaling, plays a critical role in IL-17 expression. Here, we review the current knowledge regarding mechanisms by which mTORC1 regulates IL-17 expression. mTORC1 positively modulates IL-17 expression through several pathways, i.e. STAT3, -HIF-1α, -S6K1, and -S6K2. Amino acids (AAs) also regulate IL-17 expression by being the energy source for Th17 cells, and by activating mTORC1 signaling. Altogether, the AA-mTORC1-IL-17 axis has broad therapeutic implications for IL-17-associated diseases, such as EAE, allergies, and colitis.

Glutamine promotes intestinal SIgA secretion through intestinal microbiota and IL‐13

Abstract: Scope Glutamine supplementation enhances secretory IgA (SIgA) production in the intestine, but the mechanism is largely unknown. We examined this issue using a mouse model. Methods and results In mouse model, glutamine supplementation increased both SIgA abundance in intestinal luminal contents and IgA(+) plasma cell numbers in the mouse ileum, and decreased bacterial loads in mouse mesenteric lymph nodes. Glutamine supplementation increased mouse ileal expression of cytokines associated with T cell-dependent and T cell-independent pathways of SIgA induction, including IL-5, IL-6, IL-13, transforming growth factor (TGF-β), a proliferation-inducing ligand (APRIL), B cell-activating factor (BAFF), vasoactive intestinal peptide (VIP) receptor, and retinal dehydrogenases. Injecting an IL-13 antibody during glutamine supplementation reduced J-chain expression in the mouse ileum. Glutamine supplementation increased bacterial invasion into the mouse ileal wall, while disrupting the mouse intestinal microbiota abrogated the influence of glutamine supplementation on SIgA secretion. Conclusion Glutamine supplementation appears to enhance SIgA secretion in the mouse intestine through the intestinal microbiota and subsequently through T cell-dependent and T cell-independent pathways.

Catabolism and safety of supplemental L-arginine in animals.

Abstract: L-arginine (Arg) is utilized via multiple pathways to synthesize protein and low-molecular-weight bioactive substances (e.g., nitric oxide, creatine, and polyamines) with enormous physiological importance. Furthermore, Arg regulates cell signaling pathways and gene expression to improve cardiovascular function, augment insulin sensitivity, enhance lean tissue mass, and reduce obesity in humans. Despite its versatile roles, the use of Arg as a dietary supplement is limited due to the lack of data to address concerns over its safety in humans. Data from animal studies are reviewed to assess arginine catabolism and the safety of long-term Arg supplementation. The arginase pathway was responsible for catabolism of 76-85 and 81-96 % Arg in extraintestinal tissues of pigs and rats, respectively. Dietary supplementation with Arg-HCl or the Arg base [315- and 630-mg Arg/(kg BW d) for 91 d] had no adverse effects on male or female pigs. Similarly, no safety issues were observed for male or female rats receiving supplementation with 1.8- and 3.6-g Arg/(kg BW d) for at least 91 d. Intravenous administration of Arg-HCl to gestating sheep at 81 and 180 mg Arg/(kg BW d) is safe for at least 82 and 40 d, respectively. Animals fed conventional diets can well tolerate large amounts of supplemental Arg [up to 630-mg Arg/(kg BW d) in pigs or 3.6-g Arg/(kg BW d) in rats] for 91 d, which are equivalent to 573-mg Arg/(kg BW d) for humans. Collectively, these results can help guide studies to determine the safety of long-term oral administration of Arg in humans.

Apigenin Reduces Survival of Choriocarcinoma Cells by Inducing Apoptosis via the PI3K/AKT and ERK1/2 MAPK Pathways.

Abstract: Apigenin is a flavonoid found in parsley, onions, oranges, tea, chamomile, wheat, and sprouts. It has a variety of biological properties including anti-oxidant, anti-mutagenic, anti-carcinogenic, anti-inflammatory, anti-proliferative, and anti-spasmodic effects. Based on epidemiological and case-control studies, apigenin is regarded as a novel chemotherapeutic agent against various cancer types. However, little is known about the effects of apigenin on choriocarcinoma cells. Therefore, we investigated the anti-cancer effects of apigenin on choriocarcinoma cells (JAR and JEG3) in the present study. Apigenin reduced viability and migratory properties, increased apoptosis, and suppressed mitochondrial membrane potential in both the JAR and JEG3 cells. In addition, apigenin predominantly decreased phosphorylation of AKT, P70RSK, and S6 whereas the phosphorylation of ERK1/2 and P90RSK was increased by apigenin treatment of JAR and JEG3 cells in a dose-dependent manner. Moreover, treatment of JAR and JEG3 cells with both apigenin and pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126) revealed synergistic anti-proliferative effects. Collectively, these results indicated that the apigenin is an invaluable chemopreventive agent that inhibits progression and metastasis of choriocarcinoma cells through regulation of PI3K/AKT and ERK1/2 MAPK signal transduction mechanism. J. Cell. Physiol. 231: 2690-2699, 2016. © 2016 Wiley Periodicals, Inc.

Glutamine-Induced Secretion of Intestinal Secretory Immunoglobulin A: A Mechanistic Perspective

Abstract: Secretory IgA (SIgA) is one important line of defense in intestinal mucosal surface to protect the intestinal epithelium from enteric toxins and pathogenic microorganisms. Multiple factors, such as intestinal microbiota, intestinal cytokines and nutrients, are highly involved in intestinal production of SIgA. Recently, glutamine has been shown to affect intestinal SIgA production, however, the underlying mechanism by which glutamine promotes intestinal SIgA secretion remains to know. Here, we review the current knowledge regarding glutamine in intestinal immunity, and show that glutamine enhances intestinal SIgA production may through intestinal microbiota, intestinal antigen sampling and presentation, induction pathways for SIgA production by plasma cells (both T-dependent and T-independent pathway), and even SIgA transportation. Altogether, the glutamine-intestinal SIgA axis has broad therapeutic implications for intestinal SIgA -associated diseases, such as celiac disease, allergy, and inflammatory bowel disease.

Luteolin Inhibits Proliferation and Induces Apoptosis of Human Placental Choriocarcinoma Cells by Blocking the PI3K/AKT Pathway and Regulating Sterol Regulatory Element Binding Protein Activity.

Abstract: Luteolin is a natural compound known for its anticancer effects on various human cancers by regulating signal transduction cascades. However, the effects of luteolin on human placental choriocarcinoma are not known. Results of the present study revealed that luteolin decreased viability of JAR and JEG-3 cells, which are valuable placental models, in a dose-dependent manner, and it induced apoptosis and loss of mitochondrial membrane potential in JAR and JEG-3 cells. The results also suggested that the PI3K/AKT pathway was inhibited by luteolin treatment of JAR and JEG-3 cells in a dose- and time-dependent manner. Next, we established effects of luteolin in the presence of pharmacological inhibitors of PI3K/AKT, ERK1/2 MAPK, and mTOR on proliferation of JAR and JEG-3 cells. In addition, these inhibitors were used to verify phosphorylation of AKT, GSK3beta, and ERK1/2 and to confirm mechanisms regulated by luteolin in JAR and JEG-3 cells. We also determined levels of SREBP1 and SREBP2 expression to investigate regulatory functions of luteolin in lipid metabolism in JAR and JEG-3 cells. Expression levels of both SREBP1 and SREBP2 mRNAs were significantly reduced, but only SREBP1 protein was influenced by luteolin. We compared viability of JAR and JEG-3 cells in response to luteolin alone or in combination with other chemotherapeutic drugs (etoposide, cisplatin, and paclitaxel) and found that luteolin has synergistic effects with the conventional chemotherapeutic drugs as an anticancer agent. Collectively, these results showed that luteolin plays an important role in the treatment of human choriocarcinoma cells by inhibiting the PI3K/AKT/mTOR/SREBP cascade and expression of lipogenic genes.

Curcumin Suppresses Proliferation and Migration and Induces Apoptosis on Human Placental Choriocarcinoma Cells via ERK1/2 and SAPK/JNK MAPK Signaling Pathways.

Abstract: Curcumin, a natural pigment for yellow color that originates from turmeric, is a diarylheptanoid widely studied for its anti-inflammatory, anti-angiogenic, anti-oxidant and anti-cancer effects on cells. In placental diseases including preeclampsia and preterm birth, curcumin reduces pro-inflammatory cytokines. Even though curcumin is regarded as a novel chemotherapeutic agent with strong apoptotic effects based on phenolic structure, little is known about its functional effects on choriocarcinoma. Therefore, in the present study, we investigated the chemotherapeutic effects of curcumin on choriocarcinoma cells (JAR and JEG3), which are valuable placental models. The results showed that curcumin decreased viability of choriocarcinoma cells in a dose-dependent manner. In addition, proliferative and migratory characteristics of JAR and JEG3 cells were inhibited by curcumin treatment and curcumin-induced apoptotic effects which were assessed using TUNEL and annexin V/propidium iodide (PI) staining. Moreover, curcumin decreased depolarization of mitochondrial membrane based on JC-1 staining and changed expression of apoptotic proteins. Phosphorylation of mitogen-activated protein kinases responsible for regulation of anti-cancer effects of curcumin were examined for dose- and time-dependent effects. The ERK1/2 and SAPK/JNK and their downstream molecules including P90RSK and c-Jun, respectively, were activated by curcumin. Moreover, pharmacological inhibitors of ERK1/2 (U0126) and SAPK/JNK (SP600125) suppressed ERK1/2 and SAPK/JNK activation respectively, and blockage of P38 MAPK by its inhibitor (SB203580) had a synergistic effect with curcumin. These results indicate that curcumin acts as a novel chemotherapeutic agent on human placental choriocarcinoma cells via activation of ERK1/2 and SAPK/JNK signal transduction cascades.

Fructose Synthesis and Transport at the Uterine-Placental Interface of Pigs: Cell-Specific Localization of SLC2A5, SLC2A8, and Components of the Polyol Pathway.

Abstract: The fetal fluids and uterine flushings of pigs contain higher concentrations of fructose than glucose, but fructose is not detected in maternal blood. Fructose can be synthesized from glucose via enzymes of the polyol pathway, aldose reductase (AKR1B1) and sorbitol dehydrogenase (SORD), transported across cell membranes by solute carriers SLC2A5 and SLC2A8, and converted to fructose-1-phosphate by ketohexokinase (KHK). SLC2A8, SLC2A5, AKR1B1, SORD, and KHK mRNAs and proteins were analyzed using quantitative PCR and immunohistochemistry or in situ hybridization in endometria and placentae of cyclic and pregnant gilts, cyclic gilts injected with estrogen, and ovariectomized gilts injected with progesterone. Progesterone up-regulated SLC2A8 protein in uterine luminal (LE) and glandular epithelia during the peri-implantation period, and expression became exclusively placental, chorion and blood vessels, after Day 30. P4 up-regulated SLC2A5 mRNA in uterine LE and glandular epithelia after implantation, and the chorion expressed SLC2A5 between Days 30 and 85. AKR1B1 and SORD proteins localized to uterine LE during the peri-implantation period, but expression switched to chorion by Day 20 and was maintained through Day 85. Uterine expression of AKR1B1 mRNA was down-regulated by estrogen. KHK protein localized to trophectoderm/chorion throughout gestation. These results provide evidence that components for the conversion of glucose to fructose and for fructose transport are present at the uterine-placental interface of pigs. The shift in expression from LE to chorion during pregnancy suggests free-floating conceptuses are supported by fructose synthesized by the uterus, but after implantation, the chorion becomes self-sufficient for fructose synthesis and transport.

Whole-body synthesis of l-homoarginine in pigs and rats supplemented with l-arginine

Abstract: Recent studies suggest an important role for L-homoarginine in cardiovascular, hepatic and neurological functions, as well as the regulation of glucose metabolism. However, little is known about whole-body L-homoarginine synthesis or its response to dietary L-arginine intake in animals. Four series of experiments were conducted to determine L-homoarginine synthesis and catabolism in pigs and rats. In Experiment 1, male and female pigs were fed a corn- and soybean meal-based diet supplemented with 0.0-2.42 % L-arginine-HCl. In Experiment 2, male and female rats were fed a casein-based diet, while receiving drinking water containing supplemental L-arginine-HCl to provide 0.0-3.6 g L-arginine/kg body-weight/day. In both experiments, urine collected from the animals for 24 h was analyzed for L-homoarginine and related metabolites. In Experiment 3, pigs and rats received a single oral dose of 1 or 10 mg L-homoarginine/kg body-weight, respectively, and their urine was collected for 24 h for analyses of L-homoarginine and related substances. In Experiment 4, slices of pig and rat tissues (including liver, brain, kidney, heart, and skeletal-muscle) were incubated for 1 h in Krebs-bicarbonate buffer containing 5 or 50 µM L-homoarginine. Our results indicated that: (a) animal tissues did not degrade L-homoarginine in the presence of physiological concentrations of other amino-acids; (b) 95-96 % of orally administered L-homoarginine was recovered in urine; (c) L-homoarginine was quantitatively a minor product of L-arginineg catabolism in the body; and (d) dietary L-arginine supplementation dose-dependently increased whole-body L-homoarginine synthesis. These novel findings provide a new framework for future studies of L-homoarginine metabolism and physiology in animals and humans.

Functional Roles of Fructose: Crosstalk between O-Linked Glycosylation and Phosphorylation of Akt-TSC2-MTOR Cell Signaling Cascade in Ovine Trophectoderm Cells.

Abstract: During pregnancy, the placentae of ungulate mammals (e.g., cows, sheep, and pigs) convert glucose into fructose which is the most abundant hexose sugar in fetal fluids and blood. However, the role of fructose, the most enigmatic component of carbohydrate metabolism in fetal-placental tissues, is largely ignored since it is not metabolized via the glycolytic pathway or the Krebs cycle as an energy source. Here we provided evidence for biological functions of fructose that affect proliferative behavior of the conceptus trophectoderm/chorion via activation of the Akt-TSC2-MTOR signaling cascade. The phosphorylation for activation of this cascade is mediated by O-GlcNAcylation from UDP-N-acetylglucosamine (UDP-GlcNAc), a primary product of the hexosamine biosynthesis pathway. These results reveal novel functional roles of fructose in promoting embryonic/fetal growth and development during pregnancy, and also provide new insight into understanding the relationship between excessive fructose intake and metabolic disorders.

Intracellular sources of ornithine for polyamine synthesis in endothelial cells

Abstract: Polyamines are essential for proliferation of endothelial cells (EC) and angiogenesis. This study was conducted to identify the metabolic source(s) of ornithine for polyamine synthesis in EC, using Nω-hydroxy-nor-l-arginine (Nor-NOHA, an inhibitor of arginase) and gabaculine (an inhibitor of ornithine aminotransferase; OAT). Nor-NOHA inhibited arginase with an IC50 value of 10 µM for intact EC. Nor-NOHA (0.5 mM) alone inhibited arginase activity in EC by 98 %, increased total cellular concentrations of arginine by 14 %, and decreased total cellular concentrations of ornithine, putrescine and spermidine by 17, 65 and 74 %, respectively. Arginine and glutamine contributed to 73 and 26 % of the ornithine produced by EC, respectively. Gabaculine (1 mM) alone decreased the total cellular concentrations of arginine, ornithine, putrescine, and spermidine by 14, 96, 32, and 42 %, respectively. A combination of both Nor-NOHA and gabaculine completely blocked ornithine production in EC, resulting in no detectable cellular ornithine and almost complete depletion of cellular putrescine and spermidine. Addition of 0.5 mM ornithine restored the intracellular concentrations of polyamines in EC treated with Nor-NOHA plus gabaculine, indicating that Nor-NOHA and gabaculine did not inhibit ornithine decarboxylase activity. Our results suggest that the arginase and OAT pathways are the exclusive sources of ornithine in EC when there is little extracellular ornithine and that there is intracellular compartmentalization of arginine and ornithine for endothelial synthesis of polyamines. These novel findings may have important implications for improving placental vascular growth, wound healing, and cancer therapy.

Interferon Tau Affects Mouse Intestinal Microbiota and Expression of IL-17

Abstract: This study was conducted to explore the effects of interferon tau (IFNT) on the intestinal microbiota and expression of interleukin 17 (IL-17) in the intestine of mice. IFNT supplementation increased microbial diversity in the jejunum and ileum but decreased microbial diversity in the feces. IFNT supplementation influenced the composition of the intestinal microbiota as follows: (1) decreasing the percentage of Firmicutes and increasing Bacteroidetes in the jejunum and ileum; (2) enhancing the percentage of Firmicutes but decreasing Bacteroidetes in the colon and feces; (3) decreasing Lactobacillus in the jejunum and ileum; (4) increasing the percentage of Blautia, Bacteroides, Alloprevotella, and Lactobacillus in the colon; and (5) increasing the percentage of Lactobacillus, Bacteroides, and Allobaculum, while decreasing Blautia in the feces. Also, IFNT supplementation decreased the expression of IL-17 in the intestines of normal mice and of an intestinal pathogen infected mice. In conclusion, IFNT supplementation modulates the intestinal microbiota and intestinal IL-17 expression, indicating the applicability of IFNT to treat the intestinal diseases involving IL-17 expression and microbiota.

Uterine Histotroph and Conceptus Development. II. Arginine and Secreted Phosphoprotein 1 Cooperatively Stimulate Migration and Adhesion of Ovine Trophectoderm Cells via Focal Adhesion-MTORC2 Mediated Cytoskeleton Reorganization.

Abstract: In all mammalian species, critical events, including uterine receptivity and development of the conceptus (embryo/fetus and its associated extraembryonic membranes), must be intricately orchestrated and carefully timed during the window of implantation. Otherwise, failure of conceptuses to implant is inevitable, which accounts for 50%–75% of failures to establish pregnancy. Unlike human and rodent blastocysts, the blastocysts of pigs and ruminants undergo rapid transitions from spherical to tubular and filamentous conceptuses in response to histotroph during the peri-implantation period of pregnancy. Both arginine and secreted phosphoprotein 1 (SPP1; also known as osteopontin) are multifunctional molecules that increase significantly in ovine uterine histotroph during early pregnancy; however, little is known about their relationship and synergistic effects on conceptus development. Therefore, we conducted in vitro experiments using our established ovine trophectoderm cell line (oTr1) isolated fr...

Effects of agmatine on secretion of interferon tau and catecholamines and expression of genes related to production of polyamines by ovine trophectoderm cells

Abstract: Embryonic survival requires histotrophic nutrition, including molecules secreted or transported into the uterine lumen by uterine epithelia. L-Arginine (Arg) is a common substrate for synthesis of nitric oxide, ornithine, proline, glutamate, creatinine, urea, polyamines and agmatine. Agmatine (Agm) is a product of arginine decarboxylation and it is a substrate for agmatinase for synthesis of putrescine and other polyamines in the ovine conceptus. Polyamines are essential for conceptus development. Therefore, this study compared effects of Arg and Agm on the behavior of ovine trophectoderm (oTr1) cells cultured in vitro. Arg, but not Agm, increased proliferation and migration of oTr1 cells, but neither Arg nor Agm affected cell adhesion. The total amount of IFNT in culture medium of oTr1 cells was increased by Arg, but Agm increased the IFNT production per oTr1 cell. Arg and Agm plus Arg decreased secretion of dopamine and norepinephrine by oTr1 cells. Agm upregulates expression of mRNAs SLC7A1, agmatinase and OAZ2 while the combination of Arg and Agm decreased expression of mRNAs for ODC1, SLC7A1, OAZ1 and OAZ3 by oTr1 cells. Although Agm does not stimulate proliferation, migration or adhesion of oTr1 cells or their secretion of catecholamines, Agm did increase transcription of SLC7A1, agmatinase and OAZ2 genes which would increase the capacity of oTr1 cells to produce polyamines. Collectively, our findings suggest a role for Arg and Agm in the regulation of transport of basic amino acids (including Arg), polyamine synthesis, and secretion of catecholamines by oTr1 cells.

mTOR: The Master Regulator of Conceptus Development in Response to Uterine Histotroph During Pregnancy in Ungulates

Abstract: Embryonic mortality is a major constraint to reproductive performance in all mammals. Estimates of embryonic death in most mammals, including swine, sheep, cattle, and humans, ranges from 20% to 40%, with two-thirds of the losses occurring during the peri-implantation period of pregnancy. During that stage of pregnancy, the dialog between the mammalian conceptus (embryo/fetus and associated membranes) and maternal uterus involves signaling for pregnancy recognition and maintenance of pregnancy as the stage is set for implantation and placentation that precedes fetal development. Uterine epithelial cells secrete and/or transport a wide range of molecules, including nutrients, collectively referred to as histotroph, that are transported into the fetal–placental vascular system to support growth and development of the conceptus. This review provides a framework for studies of constituents, including hexose sugars (i.e., glucose and fructose), extracellular matrix proteins (e.g., secreted phosphoprotein 1), amino acids (e.g., arginine, leucine, and glutamine), and their metabolites (e.g., nitric oxide and polyamines) that independently and cooperatively activate nutrient-sensing cell signaling pathways, particularly mechanistic target of rapamycin (mTOR), the master regulator of cell growth (proliferative growth via mTORC1 and spatial growth via mTORC2), for growth, development, and survival of conceptuses, as well as for the optimization of culture media for in vitro studies of conceptus development. Understanding the relationships between cell signaling pathways and developmental events is critical for enhancing conceptus development, implantation, and placentation, thereby increasing the probability for birth of healthy offspring.

Pregnancy and Parturition, Mammals

Abstract: Pregnancy in subprimate mammals generally requires a pregnancy recognition signal from the conceptus (embryo/fetus and associated membranes) that may be antiluteolyic. Antiluteolytic signals from the conceptuses prevent uterine production of luteolytic pulses of prostaglandin F 2α (PGF) that would cause regression of the corpus luteum and its production of progesterone. Progesterone is the hormone of pregnancy and it is required for maintenance of pregnancy in all mammals. Alternatively, a signal from the maternal anterior pituitary (e.g., prolactin) or placenta (chorionic gonadotrophin) may be a luteotrophic signal. Luteotrophic signals act directly to stimulate formation of corpora lutea and their secretion of progesterone which is required for establishment and maintenance of pregnancy, as well as maintenance of a quiescent uterus and birth of a healthy offspring. Parturition, the birth process, occurs with maturation of the hypothalamic-anterior pituitary-adrenal axis of the fetus and production of cortisol by the adrenal cortex of the fetus. Cortisol induces enzymes which metabolize progesterone to estradiol, estradiol induces enzymes that metabolize arachidonic acid to PGF and PGF causes regression of the corpus luteum. Decreasing concentrations of progesterone and increasing concentrations of estrogens increase receptors for oxytocin and PGF on the uterine myometrium and the formation of gap junctions among smooth muscles of the myometrium. This allows oxytocin and PGF to induce coordinated and strong contractions of the myometrium to expel the fetus and placenta during the birth process.