text:
MADE IN PLANO
Girl Boss 2019: Monica Shortino
by Sydni Ellis
As director of social innovation at Capital One, Monica Shortino oversees philanthropic and community strategies to lead innovation in the community. She also spends her free time helping to shape local organizations. "I have the incredible honor of impacting my community every day, through my personal life and in my career," Monica says.
Part of Monica's job at Capital One is leading the Future Edge DFW initiative, which helps Americans succeed in the digital economy. She works with area organizations including Collin College and the Boys and Girls Clubs of Collin County to build college- and career-readiness skills through science, technology, engineering, arts and math (STEAM) programming.
Currently, Monica is working to reach 10,000 high school students over the next three years with Basic TrAIning: Bot Camp, a first-of-its-kind curriculum hosted in partnership with Plano ISD and Dallas ISD that teaches artificial intelligence (AI), software development and technical problem solving. Fun fact: when this program launched back in April, it earned the Guinness World Record for largest AI programming lesson.
This passion for helping others is a personal mission for Monica. She was a first-generation college student who overcame challenges with the help of teachers, local nonprofits and church leaders who invested in her. "I wouldn't be where I am today without each of their moments of kindness and support, and I want to pay that back," Monica explained.
And she does pay it back. She has more than 10 years of service to the Junior League of Dallas and is co-chair of the community committee for Capital One's women's resource business group, EmpowHer, which mentors girls in the Young Women's Preparatory Network schools. Additionally, Monica is the former executive director for the Plano ISD Education Foundation and the former president for Plano Youth Leadership.
"As women leaders in our community, it is critical that we invest in the next generation of women leaders," Monica explained. "We must take time to mentor, provide a helping hand and cheer each other on."
Lesley Range-Stanton, executive director of communications for Plano ISD, says, "It has been my privilege to watch and to be a part of Monica's leadership journey over the past 15 years. I am so proud of all of the labels I've watched her acquire: leader, mentor, award winner, innovator, mom and now Girl Boss. But I must say, my favorite label for Monica is 'friend.'"
See all of the 2019 Girl Bosses >
Sydni Ellis
More from Sydni Ellis
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Read Our Nov/Dec Issue
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Food + Drink, News
I Love U A-Latte
ABOUT | CONTACT | COPYRIGHT © 2020 Plano Magazine, Plano, TX | 972-489-8301 |


How to Say What Are You Doing in Nepali Language: – Nepali is a language that is not too easy to learn. If your first language is not Nepali, then it takes a serious effort to learn the language. Since the script is also different including all the sounds and ways of saying things, it will not be easy to learn the language. But, if you try learning the language one phrase at a time, then it will be easier for you.
There are an only a handful of phrases that are used on a daily basis. If you just keep those phrases in mind and learn those one at a time, you can slowly learn the language well enough. Learning 'What are you doing' is one of such phrase. You can use it on a daily basis in the day to day life. So, it is a phrase that you might want to learn if you are interested in learning things in Nepali.
Before learning how to say the actual phrase, it is important to learn some of the usual expressions in Nepali. One should understand that in Nepali you have three standards of phrases for showing respect. For those who are older to you and you need to show respect, you have to say the same thing in a different way. Whereas, if the other person is younger to you, the same thing can and should be said in a much different way.
So, the same thing differs depending on the person you are talking to. If the other person is your senior, junior or your friend, the same phrase has different ways of saying it. Therefore, while learning how to say the given phrase, keep it in your mind that the same thing has three different ways of saying it depends on the person who you are referring to.
Tapain Ke Gardai Hunuhuncha? तपाई के गर्दै हुनुहुन्छ?
Tapain Ke Gardai Hununucha. This is the most respectful way of asking 'What are you doing?' It literally means what you are doing. If the person you are talking to is older than you or has a higher position and you need to respect the person, you can use this phrase to ask what he or she is doing. The phrase is similar for both male and female.
Since it means asking respectfully what he or she is doing, it can be used only for those who are higher in ranks to you or are senior to you in your relation or age. Make sure you learn how to use appropriate codes of reference according to the rankings or age of people. As we know, in English you can always refer to everyone with a simple 'you'. It is not so in Nepali. You cannot just say you, but you have different pronouns depending on the rank or age. So, follow that to make sure you do not offend the other person.
Ke Garirahanu bhayeko cha? के गरिरहनु भएको छ?
This phrase is also for those who you respect or the ones that are older than you. The phrase literally just means what you are doing. So, it is the most respectful way of asking what the other person is doing. You could use this same phrase for asking both men and women. If you ask this, as it is, you will definitely get the reply of what the other person had been doing.
Ke Gardai Ho Timi? के गर्दै हो तिमि?
Oye, Ke gariraheko? ओइ, के गरिरहेको?
Ajkal Ke ma Byasta Hunuhuncha? आजकल के मा ब्यस्त हुनुहुन्छ?
Ke Chaldai Chha? के चल्दै छ?
Nepali may be a language that's not too straightforward to find out. If your tongue isn't Nepali, then it takes a significant effort to find out the language. Since the script is additionally totally different as well as all the sounds and ways in which of claiming things, it'll not be straightforward to find out the language. But, if you are trying learning the language one phrase at a time, then it'll be easier for you.
There area unit a solely one or two of phrases that area unit utilized in a routine. If you simply keep those phrases in mind and learn those one at a time, you'll be able to slowly learn the language tolerably. Learning 'What area unit you doing' is one in all such phrase. you'll be able to use it in a very routine in the day to day life. So, it's a phrase that you simply would possibly need to find out if you're fascinated by learning things in Nepali.


Village Ancients or 'Les Anciens du Village' from Myriad Film & Research on Vimeo.
Village Ancients or 'Les Anciens du Village' looks at the rapidly evolving ways of rural life in the small village of Aragon, in the Languedoc-Roussillon area of France. Inspired by the works of Nicolas Philibert and Raymond Depardon, the film is made up of a series of vignettes about the village's inhabitants, attempting to capture a sense of Aragon in the context of its past and present.
Imogen Putler is an anthropologist and English documentary filmmaker. This film is the result of an interest in the concepts of identity and the use of social memory and narrative in the construction of identity – specifically within the small village of Aragon where she grew up. It has become a homage to the rapidly changing village of her childhood and its much-loved inhabitants.


Made a quick trip down to Wellington in Prince Edward County yesterday, meeting Craig Alexander, proprietor of the new Sybil Frank Gallery. I left him with some of my paintings and told him I always had an interest in finding representation in Beautiful Prince Edward County.
His new gallery looks great and while it is small it has huge promise. Craig is passionate about art and anyone venturing into his gallery is in for a treat. Check it out… just off main street, Wellington.
This entry was posted in In General on July 29, 2016 by peter.


MC Fitti is currently on position 2 of the German Album Charts!
I mixed the Hitsingle "Whatsapper"
MC Fitti´s Whatsapper is out now!
MC Fitti just entered the German Single Charts on #99!
i did the mastering for "30 Grad" and "Du willst so sein wie MC Fitti"
30 grad! mc fittis´summer smash hit!


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redpajama_set_name: RedPajamaGithub


What is OCR? It's the process of converting printed text into another format, like Unicode or ASCII. It's a sensitive process, and in the hands of a non-expert, you can end up with data that is either inaccurate or even unusable.
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text:
The Arthritis Foundation
James Bradberry Chooses The Arthritis Foundation For The NFL's 'My Cleats, My Cause'
The Arthritis Foundation is excited to announce that James Bradberry, NFL player for the Carolina
In my mind I'd say "Big CED is an a$$ Hole" But my heart says "It's hard to hate Big CED" So my mouth would say "Big CED is an all around good guy"!
Mitch 'Ski' DudleyLabel Marketing & SalesRED DistributionNew York, NY
"Who is BIG CED? A forward-thinking, an ambitious and energetic creative, who is unafraid of big moments and spectacles. A man who deserves his props for all the work he's done in his 20-plus-year career."
Kevin L. ClarkManaging Editor OkayplayerBrooklyn, New York
I've known Big CED for almost 20 years now, and what I like about him most is that he's still the same person now that he was back then! In this business it's hard to find genuine people, especially because the power of one's network along with success and money can often times change someone's character. But not CED, he is one of the most coolest people I know, and is also the only guy that I can call a smart-ass, but in a cool way (haha). In addition, CED is very opinionated, he's a realist, and you always know where you stand with him.
Fred TessierFounder | Managing Director, Business Operations and StrategyKeen Management Group, Inc.New York, NY
He got jokes. He is Hip Hop. He has a vision. An ageless part of this culture. His name is part of Hip Hop history and future.
Ovington PropheteCEOThe Candy Store Marketing Group (CSMG) New York, NY
professional shit talker with an ear to the streets
Shiana GregoryMedia RelationsDallas, TX
Thought provoking. Ced is one who inspires you to see different points of view. His writing is thoughtful and on purpose and causes you to think about what is being written. Although a quite giant in the writing arena, the power of his voice through words is unmatched.
Yvette N. HarrisCEOHarris Public RelationsMiami, FL
Big Ced is an entrepreneurial and social media aristocrat. But most importantly, he is a huge supporter who is always there when you need him, a wonderful friend and all around good guy!
Tamekia M. Flowers-BallOwnerEpiphany|Blue®New York, NY
Martha Munizzi Releases Two New Singles- 'He Has Won' and 'Fearless'
Tyga – Ayy Macarena
DeJ Loaf – Changes
wifisfuneral – '2 Step'
Charles Herron 'Something Like This'

redpajama_set_name: RedPajamaCommonCrawl


NIH funds over $20 million in peripheral nervous system research.
Awards from the National Institutes of Health's Common Fund are supporting research on the peripheral nervous system, in hopes of finding new ways to treat conditions such as asthma, diabetes, and nausea. The new awards total more than $20 million in fiscal year 2016, and go to 27 multidisciplinary research teams through the Stimulating Peripheral Activity to Relieve Conditions (SPARC) program. The SPARC program plans to support awards totaling approximately $238M through fiscal year 2021, pending available funds. This year's awards encompass a wide range of projects intended to resolve existing knowledge gaps and technology limitations. In addition to treatment research, other projects focus on various organ systems, including heart, lung, stomach/intestine, colon, adipose tissue, spleen, bladder, and pancreas.
SPARC focuses on understanding peripheral nerves — nerves that connect the brain and spinal cord to the rest of the body — and how their electrical signals control internal organ function. Modulation of these control signals is a potentially powerful way to treat common conditions and diseases such as rheumatoid arthritis and heart failure. Methods and medical devices that modulate peripheral nerve activity are becoming available, but more research to fully understand how these therapies act on a target organ's cells is needed. Such understanding could help to explain and resolve why a particular therapy may be effective in one individual, but not in another, thereby making these therapies effective for more people.
The 2016 awardees will work collectively as a consortium to address the goals and aims of SPARC. Biological projects will develop detailed anatomical and functional maps that illustrate how peripheral nerves control organ function, while technology development projects will create or improve tools to measure and manipulate nerve-organ interactions and isolate their functions. Additional projects include collaborations between private sector scientists and academic researchers, intended to expedite the development of new therapeutic strategies. Going forward, the SPARC program will leverage expertise from many different sources, not only academic laboratories, but also independent inventors, start-ups, small and large businesses, and international organizations. Data and tools developed in the SPARC program will be shared through a central online resource.
SPARC is funded through the NIH Common Fund and managed by the NIH Office of the Director, in partnership with the National Center for Advancing Translational Sciences, the National Institute of Biomedical Imaging and Bioengineering, the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institute of Neurological Disorders and Stroke. The fiscal year 2016 funding will be issued via NIH Other Transaction and Cooperative Agreement award mechanisms.


Ann Intern Med. 2011 Mar 15;154(6):373-83. doi: 10.7326/0003-4819-154-6-201103150-00002.
Trouillet JL1, Luyt CE, Guiguet M, Ouattara A, Vaissier E, Makri R, Nieszkowska A, Leprince P, Pavie A, Chastre J, Combes A.
Institut de Cardiologie, Hôpital de la Pitié-Salpêtriére, Assistance Publique-Hôpitaux de Paris, Université Pierre et Marie Curie, Institut National de la Santé et de la Recherche Médicale, Paris, France.
There was no difference in ventilator-free days during the first 60 days after randomization between early percutaneous tracheotomy and prolonged intubation groups (mean, 30.4 days [SD, 22.4] vs. 28.3 days [SD, 23.7], respectively; absolute difference, 2.1 days [95% CI, -4.1 to 8.3 days]) nor in 28-, 60-, or 90-day mortality rates (16% vs. 21%, 26% vs. 28%, and 30% vs. 30%, respectively). The durations of mechanical ventilation and hospitalization, as well as frequencies of ventilator-associated pneumonia and other severe infections, were also similar. However, early percutaneous tracheotomy was associated with less intravenous sedation; less time of heavy sedation; less haloperidol use for agitation, delirium, or both; fewer unscheduled extubations; better comfort and ease of care; and earlier resumption of oral nutrition. After a median follow-up of 873 days, between-group survival, psychosocial evaluations, and HRQoL were similar.


text:
Maternal supply of cysteamine alleviates oxidative stress and enhances angiogenesis in porcine placenta
Shuangbo Huang1,
Zifang Wu1,
Zihao Huang1,
Xiangyu Hao1,
Longmiao Zhang1,
Chengjun Hu1,
Jianfu Wei2,
Jinping Deng1,3 &
Chengquan Tan ORCID: orcid.org/0000-0003-1686-37781,3
Journal of Animal Science and Biotechnology volume 12, Article number: 91 (2021) Cite this article
Oxidative stress in placenta is associated with the occurrence of adverse pregnancy outcomes in sow, but there are few satisfactory treatment strategies for these conditions. This study investigated the potential of cysteamine (CS) as an antioxidant protectant for regulating the reproductive performance, redox status, and placental angiogenesis of sows.
The placental oxidative stress status and vascular density of piglets with different birth weights: < 1.0 kg (low birth weight, LBW) and 1.4–1.6 kg (normal birth weight, NBW) were evaluated, followed by allotting 84 sows to four treatments (n = 21) and feeding them with a basal diet supplemented with 0, 100, 300, or 500 mg/kg of CS from d 85 of gestation to d 21 of lactation, respectively. Placenta, serum, and colostrum samples of sows or piglets were collected, and the characteristics of sows and piglets were recorded. Furthermore, the in vivo results were validated using porcine vascular endothelial cells (PVECs).
Compared with the NBW placentae, the LBW placentae showed increased oxidative damage and were vulnerable to angiogenesis impairment. Particularly, H2O2-induced oxidative stress prompted intracellular reactive oxygen species generation and inhibited the tube formation and migration of PVECs as well as the expression of vascular endothelial growth factor-A (VEGF-A) in vitro. However, dietary CS supplementation can alleviate oxidative stress and improve the reproductive performance of sows. Specifically, compared with the control group, dietary 100 mg/kg CS could (1) decrease the stillbirth and invalid rates, and increase both the piglet birth weight in the low yield sows and the placental efficiency; (2) increase glutathione and reduce malondialdehyde in both the serum and the colostrum of sows; (3) increase the levels of total antioxidant capacity and glutathione in LBW placentae; (4) increase the vascular density, the mRNA level of VEGF-A, and the immune-staining intensity of platelet endothelial cell adhesion molecule-1 in the LBW placentae. Furthermore, the in vitro experiment indicated that CS pre-treatment could significantly reverse the NADPH oxidase 2-ROS-mediated inactivation of signal transducer and activator of transcription-3 (Stat3) signaling pathway induced by H2O2 inhibition of the proliferation, tube formation, and migration of PVECs. Meanwhile, inhibition of Stat3 significantly decreased the cell viability, tube formation and the VEGF-A protein level in CS pretreated with H2O2-cultured PVECs.
The results indicated that oxidative stress and impaired angiogenesis might contribute to the occurrence of LBW piglets during pregnancy, but CS supplementation at 100 mg/kg during late gestation and lactation of sows could alleviate oxidative stress and enhance angiogenesis in placenta, thereby increasing birth weight in low yield sows and reducing stillbirth rate. The in vitro data showed that the underlying mechanism for the positive effects of CS might be related to the activation of Stat3 in PVECs.
Pregnancy is an oxidative stress challenge for the mother, especially during the late gestation period [1], when the rapid development of the fetus increases the metabolic burdens on pregnant sows or dams, leading to elevated systemic oxidative stress [2, 3]. Increasing evidence indicates that maternal oxidative stress is associated with several adverse outcomes, such as gestational diabetes mellitus, proteinuria preeclampsia, postpartum hemorrhage, fetal death, and low birth weight [4, 5]. Thus, reducing oxidative stress is a crucial issue for improving the reproductive efficiency of both humans and mammalian animals, but its underlying mechanisms still remain elusive.
The placenta regulates fetal nutritional and hormonal support, which, to a large degree, has a direct impact on the pregnancy outcome [6]. In particular, placental blood vessels play an important role in maternal-fetal material exchange, suggesting the importance of proper placental vasculature development for fetal growth. Moreover, the placenta is highly sensitive to oxidative stress [7], which could cause vascular dysfunction in the placenta [8, 9]. Our previous studies have uncovered that increased oxidative stress induced by maternal obesity may decrease the development of placental vasculature essential for fetal growth during pregnancy [10], suggesting the involvement of placental oxidative stress in the development of adverse pregnancy outcomes. Mechanically, an increased amount of reactive oxygen species (ROS) could induce autophagy, dysfunction, and apoptotic death of vascular endothelial cells [9]. The foregoing reports indicate that targeting the placenta could be an attractive strategy for modulating oxidative stress-related pregnancy diseases.
Dietary intake of antioxidant nutrients to improve the endogenous antioxidant defense capacity has been considered a plausible way to prevent oxidative stress [6, 11, 12]. Cysteamine (CS), a precursor of glutathione (GSH), is widely used to protect a series of tissues and organs such as gastrointestinal tract [13], brain [14], and kidney [15] from oxidative stress under adverse conditions. We selected CS based on its multiple beneficial modes of action, i.e., CS could improve ileal mucosal health by regulating the oxidation status and apoptosis in finishing pigs [13], and L-cysteine, one of the downstream metabolites of CS could efficiently reduce the inflammatory response in the maternal-fetal interface and improve the placental efficiency in rats [16]. In this regard, CS appears to be a promising candidate for alleviating maternal and placental oxidative stress. However, no data are available currently regarding the effects of dietary CS supplementation during gestation and lactation on the reproductive performance and antioxidant status of sows, especially the dose-effect relationship.
In this study, we addressed the hypothesis that CS could enhance placental angiogenesis by alleviating maternal and placental oxidative stress, and ultimately benefit the survival and growth of the fetus. Thus, the purpose of this study was to evaluate the effects of CS on sows' reproductive performance, antioxidant status, and placental angiogenesis through an in vitro-in vivo method.
Animals and experimental design
This study was conducted in Guangzhou DaBeiNong Agri-animal Huabandry Science and Technology Co., Ltd. A total of 84 Landrace × York sows (parities 2–5) were allocated to four dietary treatment groups with each sow as a replicate in a completely randomized design using body weight at d 85 of gestation as a block (n = 21 per treatment). The sows in the control group (CON) received a basal gestation or lactation diet without added CS from d 85 of gestation to d 21 of lactation, while the sows in the CS group were fed a basal diet supplemented with 100, 300, or 500 mg/kg of CS (CS100, CS300, and CS500 diet). All diets were formulated to meet the National Research Council (NRC, 2012) requirements of nutrient standards for gestational and lactational sows. The ingredients and compositions of the basal diet are shown in Supplementary Table S1.
Sows were housed in individual stalls and fed twice (07:30 and 17:00) a d with a constant amount of 3 kg during late gestation. During the entire lactation period of 21 d, the piglets had no access to the sow's feed or to creep feed. Sows and piglets were given free access to water throughout the experiment. On the day of farrowing, sows were offered 2.0 kg of the lactation diet, followed by increasing the amount to 3.0 kg daily until ad libitum feeding. The reasons for the sows eliminated from this study were recorded in detail and shown in Supplementary Table S2.
Measurements of reproductive performance and sample collection
After farrowing, the number and weight of the piglets born, born alive, stillbirths and mummies were recorded, and invalid piglets included stillbirth and mummy. Piglets were weaned at d 21 of lactation.
During sow farrowing, umbilical cords were tied with a short silk line and each piglet was marked with a numbered tag to match the individual piglets with their placentae. After placental expulsion and weight recording, the placentae were collected and snap-frozen in liquid nitrogen (3 to 4 cm from the cord insertion point), and the other fresh placental tissues were immediately fixed in 4% paraformaldehyde. Placental efficiency was calculated by dividing piglet weight by placental weight [17]. In this study, the mean birth weight of the 1,181 piglets was 1.4 ± 0.22 kg (mean ± standard error). The placentae were assigned to two categories according to piglet birth weight: < 1.0 kg (low birth weight, LBW) and 1.4–1.6 kg (normal birth weight, NBW). Litter size at birth was also categorized into 2 classes based on the average number of piglets born alive per litter (15.0 piglets): low yield sow (the number of born alive piglets < 15.0) and high yield sow (the number of born alive piglets ≥15.0).
The blood samples of sows (n = 8 per group) were collected in 10 mL centrifuge tubes from the ear vein of the fasted sows at farrowing and at weaning, followed by centrifugation at 3,000×g and 4 °C for 15 min to recover the serum. The blood samples of piglets were collected from the anterior vena cava of the piglet whose body weight was closest to the average body weight of the litter at birth (NBW piglets, n = 6 per group) on the parturition day and centrifuged at 3,000×g and 4 °C for 15 min to recover the serum. At 0.5 h before the birth of the first piglet, colostrum was collected from the functional glands of each sow. Finally, these samples were stored at − 80 °C until further analysis of the oxidative parameters.
Oxidative stress parameters in serum, colostrum and placenta
The levels of total antioxidant capacity (T-AOC), glutathione (GSH), and malondialdehyde (MDA) were determined using the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's procedures.
Total protein concentrations in placenta and colostrum were measured according to the instructions of the bicinchoninic acid protein assay kit (Beyotime, Beijing, China). T-AOC, GSH, and MDA in placenta and colostrum were normalized to the total protein. T-AOC is associated with the elimination of free radicals and ROS, blocking peroxidation and thus preventing lipid peroxidation and removing catalytic metal ions, while MDA is the end product of lipid peroxidation and an excellent indicator of oxidative stress [18].
Placental vascular density
Eight sows were selected from each group, and 12 placenta samples were analyzed, including 6 NBW and 6 LBW placental samples. The number of vessels was determined via image analysis by estimating the average value of 3 slices of one placenta. Briefly, fresh placental tissues fixed in 4% paraformaldehyde were embedded in paraffin and sectioned at 5 μm thickness, followed by staining with hematoxylin and eosin. The area occupied by placental tissues was traced, and the placental vessels in these areas were also traced using a projecting microscope (Olympus CX41, Japan). For each of the 5-μm sections, the total number of vessels in the placental stroma areas were determined, then corrected with the total placental stroma areas measured (per unit area as mm2) [10].
Cell culture and treatments
Porcine vascular endothelial cells (PVECs) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in 1,640 medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2 atmosphere. The PVECs with cobblestone morphology were passaged at 90% confluence and used for experiments within five passages.
To establish in vitro oxidative stress model, H2O2 was applied to the PVECs. For the H2O2 treatment experiments, the PVECs were treated with different concentrations (0, 100, 200 or 300 μmol/L) of H2O2 for 24 h, or 200 μmol/L H2O2 for 0, 6, 12, 24 or 48 h. After treatments, the PVECs were used for subsequent analysis or treatment.
For the CS treatment experiments, the PVECs were pretreated with various concentrations of CS (0.5, 1 or 2 mmol/L) and/or 5 μmol/L stattic (a selective Stat3 inhibitor) for 2 h, and then challenged with 200 μmol/L H2O2 for 24 h. After treatments, the PVECs were used for subsequent analysis or treatment.
Cell viability assay
The PVECs (15,000 cells per well) were seeded in 96-well plates. After treatment as described above, cell viability was measured by cell counting kit-8 assay (CCK-8) (Beyotime) as instructed by the manufacturer. The absorbance of each well at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA).
Measurement of intracellular ROS generation
The accumulation of intracellular ROS was examined using the ROS assay kit (Beyotime) according to the manufacturer's instructions. Briefly, the PVECs (15,000 cells per well) were grown in a 96-well plate and subjected to different treatments as described above. After incubation with 10 μmol/L 2,7-dichlorofluorescein diacetate at 37 °C for 20 min, the fluorescence intensity of the cells was measured using the fluorescence plate reader (BD Falcon, Bedford, MA, USA) at Ex./Em. = 488/525 nm.
Scratch healing assay
The wound healing scratch assay was used to assess cell migration as previously described [19]. Briefly, cells were seeded onto a 6-well plate and cultured overnight until the formation of a confluent monolayer, followed by making a scratch wound with a 200-μL pipette tip and measuring the effects of H2O2, CS, and stattic on scratch healing at 24 h after the scratch. The images of the wounded areas were captured using an Olympus inverted microscope and quantified using the ImageJ software.
In vitro tube formation assay
Matrigel tube formation assays were used to assess the in vitro angiogenic activity of PVECs. Briefly, after treatment as described above, PVECs were seeded in 96-well plates precoated with 50 μL Matrigel (BD company, USA) at a density of 1 × 104 cells per well. After 4 h incubation, matrigel-induced morphological changes in PVECs and their tubular networks were photographed at 50 or 100× magnification for analysis using Image J software.
Trans-well migration assay
The chemotactic migration of PVECs was assayed using a trans-well chamber equipped with a polycarbonate filter with a diameter of 6.5 mm and a pore size of 8 μm. Briefly, after different treaments as described above, PVECs were suspended in 1,640 medium to a final concentration of 5 × 104 cells/ml and were then placed in the upper wells of the chamber, while the lower chamber was filled with 600 μL medium containing 10% FPS. After incubation at 37 °C for 48 h, the cell culture inserts were collected. The cells on the upper side of the filters were removed with cotton-tipped swabs, while the cells on the underside of the filters were fixed with 4% formaldehyde for 30 min, which were stained with crystal violet for 20 min and counted in five randomly chosen fields.
Quantitative real-time RT-PCR (qRT-PCR) analysis
Total RNA from placenta or PVECs was extracted with the reagent box of Total RNA Kit according to the manufacturer's instructions. The concentration of RNA was quantified using a NanoDrop® 2000 (Thermo Fisher, USA). After reverse transcription using Primer Script TM RT reagent Kit (Takara, Qingdao, China), qRT-PCR was conducted using SYBR Green on a QuantStudio 6 RealTime PCR System (Thermo Fisher, USA) under the conditions of denaturation at 95 °C for 10 min, amplification at 95 °C for 15 s and 60 °C for 1 min for 40 cycles. Each target gene was individually normalized to the reference gene β-actin by using the quantification method of 2−ΔΔct. Primers used in this study are shown in Supplemental Table S3.
Total proteins were extracted from PVECs using the protein extraction kit (Beyotime, Beijing, China) according to the manufacturer's guide. Briefly, an amount of 10 μg protein was loaded and separated by SDS–PAGE gel electrophoresis, followed by transferring the proteins onto the polyvinylidenedi fluoride membranes (Merck Millipore). After blocking with TBS/T buffer containing 5% milk, the membranes were incubated with the primary antibodies against vascular endothelial growth factor A (VEGF-A) (19003–1-AP, Proteintech, USA, 1:1,000), NADPH oxidase 2 (NOX2) (19013–1-AP, Proteintech, USA, 1:1,000), signal transducer and activator of transcription-3 (Stat3)(ab76315, Abcam, USA, 1:1,500), p-Stat3 (ab68153, Abcam, USA, 1:1,500), and β-actin (4970, CST, USA, 1:1,000). Subsequently, the membranes were incubated with appropriate HRP-conjugated anti-rabbit IgG secondary antibody (AS014, Abclonal, China, 1:5,000). Images were captured using the ChemiDoc MP system (Bio-Rad, Hercules, CA, USA), and band densities were quantified using Image Lab software (Bio-Rad, Hercules, CA, USA) and then normalized to β-actin content.
Placental tissues fixed in 4% paraformaldehyde were embedded in paraffin and sectioned at 5 μm thickness for platelet endothelial cell adhesion molecule-1 (CD31) immunofluorescence as described previously [19]. The slides were visualized under a fluorescent microscope (Nikon Eclipse C1, Tokyo, Japan). Fluorescence intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
Data are presented as mean ± SEM and were statistically analyzed using one-way ANOVA and Duncan's multiple-range test in SPSS 20.0 (SPPS Inc., Chicago). Tamhane's T2 test was used to assess variance heterogeneity. The stillbirth, LBW, and invalid rates were analyzed using the Chi-square test. Pearson's correlation coefficient was used to analyze the correlation between piglet birth weight and placental vascular density, T-AOC, and MDA. Additionally, polynomial contrasts were used to evaluate the linear and quadratic effects of CS supplementation on the various parameters measured in the sow experiment. Differences were considered significant at P < 0.05, and a tendency was considered at 0.05 ≤ P < 0.1.
Correlation between birth weight and placental characteristics of piglets
When compared with the NBW placenta, the LBW placenta showed significantly lower (P < 0.05) blood vessel density (Fig. 1A, B) and immunostaining intensity of CD31 (biomarker of the endothelial cell in small vessels) (Fig. 1C, D). Meanwhile, piglet birth weight showed a significant positive correlation with placental vascular density (R2 = 0.7146, P < 0.0001) (Fig. 1E), or T-AOC in placenta (R2 = 0.2744, P = 0.0086) (Fig. 1F), but a negative correlation with placental MDA level (R2 = 0.3700, P = 0.0016) (Fig. 1G).
The vessel density distribution in the LBW and NBW placentae. A, B The hematoxylin and eosin method was used to examine blood vessel density in the LBW and NBW placental tissues, and the black arrows indicate placental blood vessels (bar = 100 μm, n = 24). C, D CD31 immunofluorescence staining in the LBW and NBW placentae (bar = 100 μm). E Correlation between placental blood vessel density and piglet birth weight (n = 24). F Correlation between placental total antioxidant capacity (T-AOC) and piglet birth weight (n = 24). G Correlation between placental malondialdehyde (MDA) and piglet birth weight (n = 24). LBW and NBW indicate piglets with a low birth weight (LBW) (less than 1.0 kg) and normal birth weight (NBW) (1.4–1.6 kg), respectively. Data are presented as mean ± SEM. Differences between groups were considered statistically significant at ** P < 0.01
Reproductive performance
The four groups showed significant differences in sow performance (Fig. 2). When compared with the CON group, the CS100 group showed significantly lower (P < 0.05) stillbirth and invalid piglet rates, but higher (P < 0.05) placental efficiency. In addition, piglet birth weight showed significantly higher (P < 0.05) for the low yield sows of the CS100 group versus the CON group.
Characteristics of sows and piglets when supplemented with different amounts of cysteamine (CS). A, B Stillbirth and invalid piglet rates were analyzed using the Chi-square test; C Placental efficiency = piglet weight (g)/placental weight (g); D Piglet birth weight, with the number of sows being 21, 20, 19 and 19 in CON, CS100, CS300 and CS500, respectively (a-d); E, F Piglet birth weight and low birth weight (LBW, < 1.0 kg) in low yield sows (i.e., the number of piglets born alive is less than 15), with the number of sows being 11, 6, 8 and 9 in CON, CS100, CS300 and CS500, respectively (e-f); G, H Piglet birth weight and normal birth weight (NBW) in high yield sows (i.e., the number of piglets born alive is more than 15), with the number of sows being 10, 14, 11 and 10 in CON, CS100, CS300 and CS500, respectively (g-h); CON, basal diet group; CS100/300/500, basal diet supplemented with 100, 300, or 500 mg/kg of CS; Except for rates of stillbirth, invalid piglets, and LBW piglets, all values are expressed as means ± SEM. Linear (L) and quadratic (Q) effects of inclusion amounts of CS were contrasted. Different lowercase letters represent significant difference at P < 0.05
As shown in Fig. 3A-C, when compared with the CON group, the sows in the CS100 group showed significantly higher (P < 0.05) serum GSH levels at farrowing and weaning, and the sows in the CS500 group showed significantly higher (P < 0.05) serum T-AOC levels at farrowing and weaning and serum GSH levels at farrowing. For these parameters in colostrum, when compared with the CON group, the CS100 group showed a significantly higher colostrum GSH level but a lower colostrum MDA level (P < 0.05) (Fig. 3D-F). Additionally, these oxidative stress parameters in neonatal serum were also investigated (Fig. 3G-I), with a significantly higher (P < 0.05) serum GSH level for the NBW piglets in the CS500 group than in the CON group.
Oxidative stress levels of sows and piglets. A, D, and G T-AOC, total antioxidant capacity; L0, at farrowing; L21, at weaning. B, E, and H GSH, glutathione; C, F, and I MDA, malondialdehyde. CON, basal diet group; CS100/300/500, basal diet supplemented with 100, 300, or 500 mg/kg of cysteamine (CS); NBW indicate piglets with a normal birth weight (1.4–1.6 kg); Data are presented as mean ± SEM (n = 6–8). Linear (L) and quadratic (Q) effects of inclusion amounts of CS were contrasted. Different lowercase letters represent significant difference at P < 0.05
Importantly, we observed that the placentae of piglets with different birth weights react differently with maternal CS supply (Fig. 4). Compared with the CON group, the CS100 group showed significantly higher (P < 0.05) contents of T-AOC and GSH in the LBW placentae, with no difference observed in the NBW placentae among the four dietary treatments (P > 0.05).
Placental oxidative stress levels. The levels of total antioxidant capacity (T-AOC) (A, D), glutathione (GSH) (B, E), and malondialdehyde (MDA) (C, F) were normalized to the placental total protein. LBW and NBW indicate piglets with a low birth weight (LBW) (less than 1.0 kg) and normal birth weight (NBW) (1.4–1.6 kg), respectively. CON, basal diet group; CS100/300/500, basal diet supplemented with 100, 300, or 500 mg/kg of cysteamine (CS). Data are presented as mean ± SEM (n = 6). Linear (L) and quadratic (Q) effects of inclusion amounts of CS were contrasted. Different lowercase letters represent significant difference at P < 0.05
The effects of CS on placental function were further explored by analyzing the placental vascular density and the expression of angiogenesis-related genes. Figure 5A, B present the effects of CS supplementation on the placental vascular density in LBW or NBW piglets. The placental vascular density showed significantly higher (P < 0.05) for the LBW piglets of the CS100 group versus the CON group. For the NBW placentae, the CS supplementation did not significantly change the placental vascular density (P > 0.05). Immunostaining analysis also revealed higher (P < 0.05) expression levels of CD31 in the LBW placenta in the CS100 group versus the CON group (Fig. 5C, D), and similar results were also found in the mRNA level of VEGF-A (P < 0.05) (Fig. 5E).
Effects of maternal cysteamine supplementation on the vascular density of placentae. A, B The hematoxylin and eosin method was used to examine blood vessel density in placental tissues, and the black arrows indicate placental blood vessels (bar = 100 μm). C, D CD31 immunofluorescence staining in placentae (bar = 50 μm). E mRNA relative expression of vascular endothelial growth factor A (VEGF-A). CON, basal diet group; CS100/300/500, basal diet supplemented with 100, 300, or 500 mg/kg of cysteamine (CS). LBW and NBW indicate piglets with a low birth weight (LBW) (less than 1.0 kg) and normal birth weight (NBW) (1.4–1.6 kg), respectively. Data are presented as mean ± SEM (n = 6). Linear (L) and quadratic (Q) effects of inclusion amounts of CS were contrasted. Different lowercase letters represent significant difference at P < 0.05
Collectively, dietary 100 mg/kg CS could produce positive effects on the antioxidant capacity and placental angiogenesis of sows.
Oxidative stress induced by H2O2 inhibits tube formation and migration in vitro
The relationship between placental oxidative status and angiogenesis was further investigated by using PVECs to evaluate the effects of oxidative stress on angiogenesis in vitro. As shown in Fig. 6A, B, compared with the control group (0 μmol/L H2O2), the viability of PVECs was significantly decreased (P < 0.05) under H2O2 treatment in a dose- and time-dependent manner. Whether the redox state of PVECs could be changed by H2O2 was investigated by analyzing the mRNA expression of antioxidant-related genes and the endoplasmic stress markers. The mRNA expression level showed a significant decrease (P < 0.05) for GPX1, SOD1, SOD2 and CAT while a significant increase for GRP78 (P < 0.05) in a dose-dependent manner (Fig. 6C). The ROS level also increased significantly (P < 0.05) in a dose-dependent manner (Fig. 6D, E). Meanwhile, tube formation (Fig. 6F, G) and migration (Fig. 6H, I) were significantly impaired (P < 0.05) in PVECs after treatment with H2O2 for 24 h. As 200 μmol/L H2O2 treatment for 24 h could significantly decrease the viability, tube formation and migration to about 50% and change the redox state of PVECs, this concentration was used for further experiments.
Effects of oxidative stress induced by H2O2 on the tube formation and migration in porcine vascular endothelial cells (PVECs). A PVECs were treated with various concentrations of H2O2 (0, 100, 200 or 300 μmol/L) for 24 h (n = 6). B PVECs were treated with 200 μmol/L H2O2 for 0, 6, 12, 24 or 48 h. CCK8 assay was used to measure cell viability (n = 6). C The mRNA relative expression of Glutathione peroxidase 1 (GPX1), Cu/Zn superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2), catalase (CAT), activating transcription factor 4 (ATF4), and glucose-regulated protein78 (GPR78). PVECs were treated with various concentrations of H2O2 (0, 100, 200 or 300 μmol/L) for 24 h (n = 3). D, E ROS generation in PVECs treated with various concentrations of H2O2 (0, 100, 200 or 300 μmol/L) for 24 h (n = 3). F, G Scratch healing assay of migratory distance of PVECs treated with various concentrations of H2O2 (0, 100, 200 or 300 μmol/L) for 24 h (n = 3; bar = 500 μm). H, I Representative images of tube formation by PVECs treated with various concentrations of H2O2 (0, 100, 200 or 300 μmol/L) for 24 h (n = 5; bar = 100 μm). Data are presented as mean ± SEM. Different letters indicate significant differences at P < 0.05
Oxidative stress impairs the Stat3/VEGF-A pathway in PVECs
A growing body of evidence supports an important role of Stat3 in placental angiogenesis [20]. In our previous study, Stat3/VEGF-A pathway was found to be impaired in LBW placenta (Hu et al., unpublished data), leading to the question of whether H2O2-mediated angiogenesis inhibition is controlled by Stat3. After treatment with H2O2 (200 μmol/L) for 24 h, the relative mRNA expression of Stat3-regulated genes (IL6, IL8, and VEGF-A) in PVECs significantly decreased (P < 0.05), in contrast to a significant increase (P < 0.05) in the expression of NOX2 (the upstream negative regulator of Stat3) (Fig. 7A-B). Similar results were obtained from Western blotting analysis (Fig. 7C, D).
Effects of oxidative stress induced by H2O2 on the Stat3/VEGF-A pathway in porcine vascular endothelial cells (PVECs). A The mRNA expression of angiogenesis-related factors (vascular endothelial growth factor A, VEGF-A; interleukin-6/8, IL-6/8; NADPH oxidase 2, NOX2). B Schematic for the mechanism of H2O2-induced angiogenesis impairment. C, D Western blotting analysis of the expression of phospho-Stat3 (p-Stat3), NOX2, and VEGF-A. Cells were treated with 200 μmol/L H2O2 for 24 h. Data are presented as mean ± SEM (n = 3). Different letters indicate significant differences at P < 0.05
CS rescues H2O2-induced Stat3 signaling pathway activity
The effects of CS on the ROS level and endothelial dysfunction in H2O2-induced PVECs were also evaluated. As shown in Fig. 8A, E, the ROS level induced by H2O2 was reduced in the cells pretreated with CS (P < 0.05). In addition, the impaired migration by scratch healing assay (P < 0.05) (Fig. 8B, F) or by trans-well assay (P < 0.05) (Fig. 8C, G), and tube formation (P < 0.05) (Fig. 8D, H) induced by H2O2 were partially recovered in PVECs pretreated with CS (P < 0.05). Similarly, H2O2 resulted in a significant decrease in cell viability, but CS supplementation could alleviate the negative effect of H2O2 on cell viability (P < 0.05) (Fig. 8I).
Cysteamine (CS) pretreatment attenuates the effects of H2O2 on angiogenesis. A, E The levels of ROS. PVECs were pretreated with various concentrations of CS (0.5, 1 or 2 mmol/L) for 2 h and then treated with 200 μmol/L H2O2 for 24 h (n = 6; bar = 100 μm). B, F Scratch healing assay of migratory distance. PVECs were pretreated with various concentrations of CS (0.5, 1 or 2 mmol/L) for 2 h and then treated with 200 μmol/L H2O2 for 24 h (n = 3; bar = 500 μm). C, G Trans-well migration assay of the migratory number of PVECs. After different treatments as described above, PVECs were added to the upper chamber of a trans-well and incubated for 48 h, followed by quantifying PVECs that invaded through the chamber (n = 3; bar = 500 μm). D, H Representative images of tube formation of PVECs after different treatments as described above (n = 5; bar = 100 μm). I CCK8 assay was used to measure cell viability after different treatments as described above (n = 6). Data are presented as mean ± SEM (n = 3). Different letters indicate significant differences at P < 0.05
Previous studies have shown that CS could activate Stat3 signaling and provide the critical protection required for the survival of intestinal epithelial cells in vivo [21]. Therefore, we hypothesized that Stat3/VEGF-A might mediate the enhanced endothelial function in oxidative stress with CS supplementation. The data showed that CS could largely reverse the H2O2-downregulated Stat3/VEGF-A activity (P < 0.05) (Fig. 9A, B). Furthermore, pretreatment with stattic, a selective Stat3 inhibitor, abrogated the effects of CS-mediated pro-angiogenesis (P < 0.05) (Fig. 9C, D) and pro-viability (P < 0.05) (Fig. 9E) against H2O2 in PVECs. Meanwhile, inhibition of Stat3 significantly decreased (P < 0.05) the cell viability and the VEGF-A protein level in CS pretreated with H2O2-cultured PVECs (Fig. 9F, G).
Cysteamine (CS) pretreatment prolongs the phosphorylation of Stat3 in H2O2-treated PVECs. A, B Western blotting analysis of the expression of Phospho-Stat3 (p-Stat3), NADPH oxidase 2 (NOX2), and vascular endothelial growth factor A (VEGF-A). Cells were pretreated with various concentrations of cysteamine (0.5, 1 or 2 mmol/L CS) for 2 h, and then challenged with H2O2 (200 μmol/L) for 24 h (n = 3). C, D Representative images of tube formation by PVECs, pretreated with CS (0.5 mmol/L) and/or inhibitors of Stat3 (5 μmol/L stattic) for 2 h, and then challenged with H2O2 (200 μmol/L) for 24 h (n = 5; bar = 100 μm)5. E CCK8 assay was used to measure cell viability after different treatments as described above (n = 6). F, G Western blotting analysis of the expression of p-Stat3 and VEGF-A (n = 3). Data are presented as mean ± SEM. Different letters indicate significant differences at P < 0.05
The above data confirmed that the Stat3/VEGF-A signaling pathway is invovled in the endothelial protection of CS against oxidative stress.
Previous studies have shown that increased oxidative stress in placenta is associated with the occurrence of adverse pregnancy outcomes, including intrauterine growth restriction and stillbirth [22, 23], suggesting that oxidative stress might contribute to the occurrence of low birth weight and stillbirth. However, the molecular mechanism underlying this link remains unclear. In pigs, high occurrence of intrauterine growth restriction and stillbirth is widely reported [24]. Additionally, pigs are one of the animals most commonly used in biomedical studies on human pregnancy, primarily because of their physiological similarities to humans [25, 26]. The objective of this study was to investigate whether maternal CS supplementation during late gestation improves pregnancy outcomes by examining the changes in antioxidant status and placental angiogenesis. Our study demonstrated that the placentae for LBW neonates were vulnerable to oxidative stress, which might contribute to the occurrence of LBW piglets during pregnancy. However, CS supplementation was shown to have great potential to improve pregnancy outcomes in sows, including birth weight and stillbirth, by reducing oxidative stress and enhancing angiogenesis in placenta.
Placenta plays an important role in fetal growth [27]. Thus, differences in the placental oxidation parameters of piglets with different birth weights were first determined in the present study. We found that birth weight was positively correlated with T-AOC, but negatively correlated with MDA, which was in line with Takagi et al., who reported that oxidative DNA damage was increased in intrauterine growth- restricted placenta and fetus [7]. Excessive ROS could be produced at certain windows in placental development and in some pathological pregnancies, such as those complicated by preeclampsia and/or intrauterine growth restriction, overpowering antioxidant defenses with deleterious outcome [28, 29]. Placental blood vessels are important for fetal growth and development [30], and increased oxidative stress might have a significant effect on placental function, including the proliferation and differentiation of trophoblast cells and vascular reactivity [8, 31]. Likewise, in this study, the LBW placentae showed increased oxidative damage and were vulnerable to angiogenesis impairment. Particularly, oxidative stress induced by H2O2 (a major type of ROS) prompted intracellular ROS generation and inhibited the tube formation and migration of PVECs as well as the expression of VEGF-A (a major driver for blood vessel formation) in vitro. These findings suggest that birth weight is associated with placental redox state, angiogenesis, or both.
Increased oxidative stress during late gestation could lead to a greater percentage of stillbirth [32, 33], which is a substantial cause of economic loss in livestock [34]. Our previous data indicated that alleviating serum oxidative stress at day 109 of gestation contributed to reducing the stillbirth of sows [18]. In this study, CS supply during late gestation was shown to improve the pregnancy outcomes, including birth weight and stillbirth in sows. To test whether the mechanism of CS in improving pregnancy outcomes is related to oxidative stress, we analyzed the effect of dietary CS supplementation on the related parameters in serum and colostrum, and found dietary CS supplementation could partially improve the antioxidant status of sows and their offspring as well as reduce the oxidative stress parameters. Meanwhile, maternal CS supplementation was also shown to partially increase the antioxidant status of placentae for LBW piglets. CS is a known antioxidant and anti-inflammatory agent [35], and previous studies have proposed a number of mechanisms for its protection against oxidative stress. Firstly, CS was reported as a contributor to the cellular redox state of hepatocytes by acting through sulfhydryl-disulfide exchange reactions in cells [35]. Secondly, CS could inhibit nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 inflammasome by metabolizing cysteine, thereby improving the redox status of maternal-placental interface in sows [11]. Moreover, when used at low concentrations, CS could promote the transport of cysteine into cells, which can be further used to synthesize GSH (one of the most potent intracellular antioxidants) and influence the cellular redox homeostasis [36]. GSH status could reflect the antioxidant capacity of developing embryos. Previous studies have shown that GSH could reduce the formation of ROS and ultimately protect embryos against oxidative stress [37, 38]. In guinea pig, supplementation with GSH precursor N-acetylcysteine was shown to normalize the endothelial function in intrauterine growth-restricted placenta and fetus, thereby normalizing fetal growth [39]. In the present study, the GSH levels were elevated in the LBW placentae of the CS100 group, which benefits the growth and survival of embryo and protects the fetus against ROS damage. In addition, we note that CS supplementation increased the birth weight of piglets only in sows with a low number of piglets, which may be related to their limited uterine capacity in sows with a high number of piglets. Previous reports suggest that at moderate intrauterine crowding, litter size reaches a peak, and further crowding beyond this point could reduce the number of viable embryos/fetuses and weight accumulation, probably by reducing the number of embryos and weight accumulation able to obtain sufficient uterine space for survival due to intrauterine competition for space among embryos [40]. One point we must emphasize is that the litter size is much higher for high yield sows (average 20) than low yield sows (average 13) in this study, which may mask the beneficial effects of CS on high yield sows. The potential mechanism needs to be elucidated in further studies.
Previous reports on the effects of dietary CS levels on redox status and growth performance of animal models, including finishing pigs, sheep and rats, are inconsistent with each other [41]. For instance, previous studies in pregnant rats have shown an increased risk of oxidative stress and IUGR when high doses of CS (150 mg/kg) are applied [41], suggesting the importance of CS dose. In the present study, another important finding is that the dose-effect relationship between CS supplementation and the redox state and pregnancy outcomes is not linear. Compared with the higher dose, the minimum dose (in vivo and in vitro doses were 100 mg/kg and 500 μmol/L, respectively) was shown as the suitable supplemental dose. A possible explanation is that when used at higher doses, cysteamine oxidation in the presence of transition metals generates H2O2 molecules, thus causing oxidative stress. In addition, high doses of CS diminish the activity of glutathione peroxidase, the enzyme that catalyzes the oxidation of glutathione to its disulfide [42]. Thus, divergent results of studies using cysteamine could frequently be explained by the antioxidant effect of the drug, which could be counteracted by its direct toxicity when higher doses are used [36]. However, the potential mechanism needs to be revealed in further studies.
The dense blood vessel network in the placenta is responsible for the exchange of respiratory gases, nutrients, and wastes between mother and fetus throughout pregnancy, which is essential for normal fetal growth [43]. Throughout gestation, the vasculature of the placenta is constantly evolving to accommodate the mounting demands of the fetus and could be directly influenced by a number of exogenous factors, such as maternal diet, smoking, oxidative stress, and medication use [44]. Therefore, the effect of CS on the placental vessel density in piglets with different body weights was evaluated in the present study, and 100 mg/kg CS dietary supplementation was found to increase the placental vessel density in LBW piglets. CD31 is a biomarker of endothelial cells in blood vessels, and VEGF-A is a major driver of blood vessel formation [19]. The increased CD31 immunofluorescence intensity and VEGF-A mRNA level further demonstrated that the placental vessel density was higher in the CS100 group than in the other three groups. It is worth noting that changes in placental vessel density were consistent with oxidative stress levels only in the LBW placentae among the four groups, further implying that oxidative stress might play an important role in placental angiogenesis. Another key factor for efficient nutrient exchange is placental efficiency [17]. Variations in placental efficiency, a measure of grams of fetus produced per gram of placenta, were initially studied between swine breeds, where increased placental efficiency was found to be associated with larger litters. For instance, when compared to Yorkshire pigs, Meishan pigs, which are known for large litter sizes, have smaller placentae, but higher vascularity [45]. These observations seem logical in that proper placental vascularization facilitates the efficient exchange of nutrients, implying the importance of improving overall placental efficiency [46], which might also help explain the increased placental efficiency in the CS100 group. Overall, maternal CS supply could decrease the oxidative stress level, thus enhancing angiogenesis in placentae.
Previous studies have reported Stat3 as an important regulator of the adaptive response to oxidative stress in the placenta, which might be related to the increased oxidative stress and the altered trophoblast invasion and placental angiogenesis [44]. The expression of Stat3 and Stat3-mediated genes was shown to be downregulated in our recent study (unpublished data). In the present study, Stat3 targeting genes (VEGF-A, IL-6, and IL-8) and upstream negative regulators (NOX2) were used to validate the in vitro modulation of H2O2. The data of our study and others have demonstrated that H2O2 inhibits the proliferation and migration of PVECs by upregulating NOX2-ROS-mediated inactivation of Stat3 signaling pathway in vitro [47], indicating that Stat3 might play an important role in placental angiogenesis in porcine. Previous studies have also shown that CS could activate Stat3 signaling and provide the critical protection required for the survival of intestinal epithelial cells in vivo [21]. Here, we found a novel function of CS, modulating the repair of H2O2-induced PVECs oxidative stress through the Stat3 signaling pathway. The pretreatment of PVECs with statttic (a selective Stat3 inhibitor) and CS failed to prevent the decrease of VEGF-A protein expression and tubification ability induced by H2O2, strongly suggesting that Stat3 mediates the endothelial protective effect of CS in vitro.
In this study, oxidative stress and impaired angiogenesis were shown to contribute to the occurrence of low-birth-weight piglets during pregnancy, but maternal CS supply at 100 mg/kg during late gestation and lactation of sows could alleviate oxidative stress and enhance angiogenesis in LBW placentae, thereby enhancing pregnancy outcomes, including increased birth weight in low yield sows and reduced stillbirth. The in vitro data showed that the underlying mechanism for the positive effects of CS might be related to Stat3 activation in PVECs.
The datasets produced and/or analyzed during the current study are available from the corresponding author on reasonable request.
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The authors would like to thank the Guangzhou DaBeiNong Agri-animal Huabandry Science and Technology Co., Ltd. with limited liability for animal management.
This project was funded by Project of National Natural Science Foundation of China (No. 31872985 and 31790411) and Natural Science Foundation of Guangdong Province (No. 2021A1515012116).
Guangdong Provincial Key Laboratory of Animal Nutrition Control, National Engineering Research Center for Breeding Swine Industry, Institute of Subtropical Animal Nutrition and Feed, College of Animal Science, South China Agricultural University, Guangzhou, 510642, Guangdong, China
Shuangbo Huang, Zifang Wu, Zihao Huang, Xiangyu Hao, Longmiao Zhang, Chengjun Hu, Jinping Deng & Chengquan Tan
Guangzhou DaBeiNong Agri-animal Huabandry Science and Technology Co., Ltd., Guangzhou, 510642, Guangdong, China
Jianfu Wei
Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, 510642, Guangdong, China
Jinping Deng & Chengquan Tan
Shuangbo Huang
Zifang Wu
Zihao Huang
Xiangyu Hao
Longmiao Zhang
Chengjun Hu
Jinping Deng
Chengquan Tan
The author contributions are as follows: C.Q.Tan, J.P. Deng and S.B. Huang conceived and designed the experimental plan. S.B. Huang, Z.F. Wu, X.Y. Hao and Z.H. Huang were involved in the animal experiments, analysis and data collection. S.B. Huang wrote the manuscript. C.J. Hu, J.F. Wei and L.M. Zhang assisted with the manuscript preparation. The author(s) read and approved the final manuscript.
Correspondence to Jinping Deng or Chengquan Tan.
All procedures involving animals were approved by the Animal Ethics Committee of South China Agricultural University and followed the Guidelines for the Care and Use of Laboratory Animals of South China Agricultural University (Guangzhou, China).
There were no conflicts of interest.
Additional file 1: Supplementary Table S1.
Composition and nutritional level of gestation and lactation basal diets (Air-Dry Basis, %).
The number of sows during the experimental period.
Primer sequences used for real-time PCR.
Huang, S., Wu, Z., Huang, Z. et al. Maternal supply of cysteamine alleviates oxidative stress and enhances angiogenesis in porcine placenta. J Animal Sci Biotechnol 12, 91 (2021). https://doi.org/10.1186/s40104-021-00609-8
Cysteamine


Flagler County has furnished the state of Florida with its initial "individual" damage assessment reports from Hurricane Matthew in the amount $72,820,117.
State of Florida Department of Emergency Management will travel to Flagler County to perform its own assessment to ensure they are in agreement with our initial individual numbers.
Estimated residential damage totaled $66,124,679.
So far, 11 homes in Flagler County were destroyed. Another 164 received major damage, which – by FEMA definition – means that repairs are possible, but not within 30 days. These numbers will likely change.
Homes that received minor damage – that which can be repaired in fewer than 30 days number 294. In total there were 1,276 with properties that were "affected," which can include things like a fallen tree leaning on a house that has not penetrated it.
Unincorporated Flagler County suffered $49,659,830 in estimated total damages – and $48,033,938 in residential damages. That includes 462 affected single-family homes, 242 with minor damage, 159 with major damage, and six that were destroyed.
Flagler Beach had total estimated total damages of $15,178,829 – and $13,166,109 residential. Five homes were destroyed, another five suffered major damage, 27 minor damage and 558 affected residential homes were affected.
One restaurant suffered major damage, one with minor damage and another five were affected. Damage to restaurants is estimated to be $426,402 and hotels suffered another $414,302 in damages.
Total estimated damages in Beverly Beach are estimated to be $4,001,517. Marineland suffered an estimated $135,515 in damages.
Palm Coast total damages are estimated to be $3,585,412. Total damages in Bunnell are estimated at $259,014.
Flagler County Continues to operate a call center from 6 a.m. to 8 p.m. at 386-586-5111. The Red Cross has established a Hurricane Matthew hotline at 800-768-8048 to provide information about the resources available in every county.
The Flagler County Sheriff's office is investigating at least 14 residential and three commercial burglaries that occurred as Hurricane Matthew came through and the two days that followed. In most cases, the burglars targeted homes or businesses where the owners had evacuated.
Guns, jewelry, electronics, cash and even a vehicle were stolen.
In two cases, residents lost their unsecured generators when someone stole them from their property – one, valued at $1,200, had just been turned off for the night.
The main point of entry in the home burglaries was kicking in or damaging front doors or shattering back sliding-glass doors. The residential burglaries occurred in Palm Coast and Bunnell.
Robert Weidig Gunsmith, 15 Utility Drive, Palm Coast. 41 firearms stolen.
Cricket Wireless, 1499 Palm Coast Parkway N.W., Palm Coast. Two deposit bags and petty cash were stolen.
Mobil gas station, 5935 E. S.R. 100, Palm Coast. Still under investigation.
The Sheriff's Office is asking for the public's help solving the burglaries. Observant and vigilant neighbors can provide a good description of a suspect or suspects or vehicles involved can be a great help. Detectives said if you see something suspicious, it's important to say something.
Anyone with information that can help the Sheriff's Office is asked to call 386-313-4911. If you prefer to remain anonymous, you can call Crime Stoppers of Northeast Florida at 888-277-8477. Tipsters may be eligible for a reward of up to $1,000.
Gov. Rick Scott and Rep. John Mica toured the damage of a one-mile stretch of State Road A1A in Flagler Beach where a large portion of the road was washed away when Hurricane Matthew lashed the area.
They said the next step is starting a restoration project.
Scott and Mica said they can't say for sure when the project will start. They did say it'll be a project that will take several months. Mica said the damage to A1A caused by Hurricane Matthew is the worst to the state's transportation system.
Looking at the damage, Scott and Mica determined that the cost to fix the road will be very high.
Scott said the state will be working with the federal government for funds, as well as the Army Corp of Engineers. Both men said they will work on preparing contracts to begin the work, then work on a preliminary design.
"We're going to work with the governor and the mayor and others and the county," Mica said. "This would bankrupt Flagler Beach and the county to restore this."
Another topic that came up at the news conference was the safety of the beaches.
Flagler County's beaches will be closed until further notice because of safety issues.
That debris includes wood from the pier that was partially destroyed, roofing nails, and both metal and wood from the dune walkovers, which have also been left unsafe for use.
The Flagler County Supervisor of Elections offices reopened Monday.
The Palm Coast Branch of the Flagler County Public Library will be open from noon until 5 p.m., with limited services available. There will be a limited supply of water and ready-to-eat meals distributed from this location.
For general questions, use the citizen information call-in line at 386-586-5111 or www.flagleremegency.com.
Hours of operation are changing to 6 a.m. to 8 p.m.
Flagler County will open an assistance center on Wednesday to help residents get information and some resources for their needs in the wake of Hurricane Matthew. The center is ahead of a potential FEMA Disaster Center.
The center will be housed at the Palm Coast Branch Library at 2500 Palm Coast Parkway N.W., and it will be open from 9 a.m. to 6 p.m. beginning Wednesday.
Multiple agencies will be located at the Assistance Center, including United Way, Stewart-Marchman-Act Behavioral Healthcare, American Red Cross, Salvation Army, Flagler County Health Department, Flagler County Human Services, Flagler County Homebuilder's Association, Department of Children and Families, FEMA (once they arrive) and Verizon with two charging stations.
"There are already people at the library charging their phones," said Flagler County IT Director Jarrod Shupe. "It is really great that they provided us with these charging stations.
Small satellite facilities will be set up at the Adult Education Center at 5633 N. State Road A1A in Hammock, and at the Hidden Trails Community Center at 6108 Mahogany Blvd. in Daytona North. Residents will be pre-screened for necessary services and provide counseling.
Flagler County will be waiving some building permit fees to fast-track the permitting process for those whose properties were damaged by Hurricane Matthew.
The county will waive fees for permits that are related to hurricane damage until December 5.
Contractors must be registered with Contractor Licensing to work in Flagler County. Proof of License and Liability Insurance is required.
Residents and business owners who have questions about either permits or inspections should call the Flagler County Building Department at 386-313-4002.
Flagler County Continues to operate a call center from 6 a.m. to 8 p.m. at 386-586-5111.
The Red Cross has established a Hurricane Matthew hotline at 800-768-8048 to provide information about the resources available in every county.
There are several spots throughout Flagler County where residents can get a meal on Tuesday, and the American Red Cross has four roving vehicles distributing meals.
The Salvation Army is near the Bunnell Housing Authority, 414 Bacher Street, Bunnell, noon until 6 p.m.
The Salvation Army is in Beverly Beach providing meals at 2814 N. Oceanshore Blvd., near Surfside Estates at noon to 6 p.m.
Red Cross is providing meals at the Adult Education Center parking lot, 5633 N. Highway A1A, Hammock, at noon and 5 p.m.
Red Cross is at Flagler County Main Branch Library parking lot, 2500 Palm Coast Pkwy NW, Palm Coast, providing meals at noon and 5 p.m.
Red Cross is at Town Center in Palm Coast (where the food trucks stage), 975 Central Avenue, at noon and 5 p.m.
Red Cross is providing meals at Hidden Trails Community Center parking lot, 6108 Mahogany Blvd., at noon and 5 p.m.
Four roving vehicles are circulating throughout Flagler County.
The "Point of Distribution" for food and water at the Flagler County Executive Airport will close at 4 p.m. Tuesday.
Supervisor Jacob Oliva reported that school staff is working hard to clean all of its facilities so classes for students can be resumed.
Church on the Rock is at 2200 N. State Street, Bunnell. The phone number is 386-437-9915.
Most of State Road A1A in Flagler County is undrivable, with long sections of the highway washed out by the storm surge.
"It's unbelievable what nature can do," storm survivor Cheryl Combee said. "We're blessed. I feel very blessed by God."
Combee said she realized she was lucky after choosing not to evacuate from Beachside because of her cats. She said she wouldn't stay behind again.
"It was very scary," she said. "You don't know what's going to happen. You're risking your life."
The damage was heaviest in the northern section, in Marineland Acres. There was damage to the Flagler Beach Pier, but there's no specific assessment yet.
A husband and wife had to be rescued from their home, but no deaths or injuries were reported.
Inoperable stop lights must be treated as four-way stop signs.
"We cannot have people going through intersections without checking for traffic coming from the opposite directions," said Flagler Beach Police Chief Matt Doughney.
For general questions, please use the citizen information call-in line at 386-586-5111. People are answering calls 24 hours a day to help keep the 911 lines free for emergencies.
The damage in Surfside Estates was overwhelming.
There are a few hundred homes in the neighborhood and large parts of roofs were torn off; the side paneling to homes was found down the street.
About every other home has some sort of damage to it, most of it pretty significant, such as screen porches blown away and awnings ripped off. Many people said it looked like a tornado touched down.
"It was heartbreaking coming through here and seeing some of our neighbors' homes are gone," Therese Morgan said.
Morgan evacuated to Tampa for the last three days with her family, unsure what she was going to come back to. Dozens of neighbors returned to find only half of their homes intact.
Morgan said it looked as if the winds played hopscotch with the hundreds of houses throughout the neighborhood.
"Driving around today, it would skip a couple of homes and then the next home, the fronts are demolished, the roofs are gone, you can see through the homes," Morgan said.
Robert Hudson's home was left untouched. His next door neighbor's carport was ripped off.
"It could have been a lot worse if that hurricane would have been inland a little more," Hudson said. "If it had been 150 miles an hour, I don't think we would even be here right now."
Street after street, it was the same scenario. Panels and awnings torn off, pieces of glass and metal scattered through yards, the sides of some houses leveled.
Ed Noack said he imagined the hurricane was going to be bad, but seeing the reality of the force of its winds is unbelievable.
The neighbors in the 55-and-older community are pulling together to get homes rebuilt now and in the coming days.
City of Palm Coast telephones came back up Tuesday morning. All city departments, including the Customer Service and Building Divisions, are now available at their regular phone numbers.
Customer Service can be reached at 386-986-2360. Representatives can assist with any calls about water and sewer (wastewater) service, pep tank alarms, and questions about debris pickup.
The Building Division can be reached at 386-986-3780 for permitting questions or to discuss whether the contractor you're hiring has the right license.
While it appears that Hurricane Matthew will not rear its head again as officials once feared, it does appear that Flagler County will experience breezy conditions beginning Monday.
Conditions are predicted to be worse along the coast than inland.
Because damage assessments and cleanup operations will take time, as well as the continued need of shelter space for residents, all Flagler County schools will remain closed Monday and Tuesday, Superintendent Jacob Oliva said Saturday.
School officials said Tuesday night that a fake social media account was putting out false information.
Classes are still scheduled to resume Wednesday, Oct. 12, Flagler County Schools spokesman Jason Wheeler said.
Flagler County schools need to make up five days following hurricanes Hermine ad Matthew. The first of the hurricane make up days will be Friday. It was originally scheduled to be a teacher work day. Additional make up days will be announced at a later date.
Flagler County officials are urging residents to follow safety tips around floodwater.
Clean and dry everything that got wet, as floodwaters pick up sewage and chemicals from roads and commercial buildings.
Throw out spoiled and cosmetics and medications that have gotten wet. They are a health hazard. Contact the Health Department at 386-437-7358 to dispose of medications.
Do not let children play in or around high water, storm drains or ditches. Besides drowning risks, the water could be contaminated. Additionally, debris and other hazards may not be visible beneath the surface.
Look before you step. Floors and stairs may be covered with slippery mud or other hazardous materials.
Remember that there are animal hazards such as snakes, rodents (and other small animals), fire ants and scorpions. Any or all of these creatures could have entered homes and buildings looking for shelter.


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Robert Panatieri
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Robert Panatieri, 60, passed away at his home in Monroe Township Monday December 06, 2021, surrounded by his loving family.
Robert was born in New Brunswick to Sylvester and Maria Panatieri and was raised in Piscataway Township.
Robert met and married his wife of twenty-eight years, Denise (Duh) Panatieri and started their life together and then settled in Monroe Township. Robert worked for the Hilton Newark Airport as a lead engineer for the last twenty-eight years.
Robert was a family first man. He enjoyed traveling, especially Florida and its warm weather and beaches. He was a Dolphins and New York Giants fan but among all those things he most enjoyed being with and around his family and friends.
Robert is predeceased by his parents, Sylvester and Maria Panatieri.
He is survived by his loving wife, Denise Panatieri, his brothers; Gene Panatieri of Wilkes-Barre PA, Marco and Susanna Panatieri of Neshanic Station, Christopher Panatieri and Tracy Wall of Neshanic Station. He also leaves behind his sisters, Denise Smith of Monroe Township, as well as Brenda and Dan Gallagher of Warren. His Aunt and Uncle; Frances Mattiucci of Hudson, PA, Gene and Pauline Panatieri of Lafin, PA and a host of nieces and nephews.
Robert will be sadly missed by his Father and Mother-in-law, Frank and Kathy Duh of Old Bridge as well as his brother and sister in laws; David and Lauren Duh of Shepherdstown, WV, Dan and Cynthia Duh of South Bound Brook, Don and Michele Duh of South Bound Brook, Darlene and Jack Coughlin of Monroe Township, Deanne and Art Illenburger of New Windsor, NY and his best friend/brother Peter Stamile of Monroe NJ as well as his many friends and family in Italy. He will truly be missed.
Visitation will be held at the M. David DeMarco Funeral Home, 205 Rhode Hall Road, Monroe Township on Sunday December 12, 2021, from 2:00 pm – 4:00 pm and then again from 6:00 pm – 8:00 pm.
Another second visitation will be held at the funeral home on Monday, December 13, 2021, from 8:30 am – 9:30 am
A funeral liturgy will be held at Nativity of Lord Catholic Church, 185 Applegarth Road, Monroe Township on Monday, December 13, 2021, beginning at 10:00 am.
Entombment will immediately follow mass at Resurrection Cemetery, 899 East Lincoln Avenue, Piscataway, NJ 08854.
In Lieu of Flowers memorial contributions may be made to Steeplechase Cancer Center at Robert Wood Johnson University Hospital Somerset. Checks can be mailed to 110 Rehill Avenue Somerville, NJ 08876 or visit https://www.rwjbh.org/rwj-university-hospital-somerset/giving/give-now/ please pick designation – Cancer Center.
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M. David DeMarco Funeral Home Inc.
205 Rhode Hall Road
Monroe Township, New Jersey, United States
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Nativity Of Our Lord RC Church
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