Another promising method is based on combinatorial antigen recognition with balanced signaling in which nonspecific antigens A and B can both express on tumor cells while normal ones only express one of them.49 The gene-modified T cells can express both a first-generation CAR with CD3ζ as antigen A recognition element and a third-generation CAR with chimeric costimulatory receptors (CCRs) like CD28/CD137 as antigen B recognition element. When the T cells meet the normal cells that express only antigens A or B, the former cells can only provide a low-affinitive activating signals from CD3ζ without costimulatory signals from CCR while the latter cells just provide costimulatory signals without T cells activating signals from CD3ζ, which in any case provide insufficient T cells activating signals and thus protect normal cells. However, if recognize tumor cells that express both antigen A and B, the activating signals from A and the costimulatory signals from B can work together to active T cells and therefore release granzyme and perforin to kill the tumor cells. The key point of this method is the diversity of affinity in which CD3ζ holds a lower affinity with its target antigen while CCR holds a high affinity, only when the CD3ζ and CCR are binding together with their target antigens can the T cells be activated.
Sutton Parks was. Now the owner of an office cleaning business in Franklin, Tennessee, Parks lived in his 1993 Chrysler New Yorker for much of 2005 when he was 37 years old and was evicted from his foreclosed home in Spring Hill, Tennessee. He had lost his job as a computer operator, and his life took an abrupt turn that left him living in his vehicle.
the police have to respond to a call, that is check you out, but they do not have to make you leave, unless the owner called them or it is really illegal. sometimes police will tell me, “just leave if someone tells you to.” or they will ask me if there is anything I need, like a shelter or social service. I was humored when a Colorado officer gave me his business card.
Don’t underestimate warning lights: Your vehicle is equipped with various warning lights to indicate when there’s a problem. Unfortunately, your liver’s warning lights may not be as clear. Signs of liver disease can often be misinterpreted, such as flu-like symptoms, fatigue, or lack of appetite. If you have any of these symptoms and/or abdominal pain, dark urine, or jaundice, consult your doctor.
How I spend my time I wake up whenever between 05:00 and 09:00, hit the gym (about 8 blocks from closest sleeping spot, 2 miles from farthest sleeping spot) for about 45 minutes or so, shower shave change cloths and I am gone again. There is a unemployement/welfare building about .5 miles from gym. I dont collect unemployment, or any other government money or subsidies (dont qualify for any), a few times a week I go there after the gym to kill a few hours using there free computers to check my email and look for work online. They did an awsum job helping me put together a great resume, perhaps the first gov. assistance I have ever received in my life.
I have a solar panel set up myself and I looked at the Yeti 400 at Goal Zero Solar website. You could really save a lot of money getting some solar panels and charge controller from Renogy. They are also online. You have to purchase your own battery and also an inverter, but you can get a lot more power for a lot less money. I can give you the details of my set up if you like. Camping in this country is designed and almost designated for professional people who have money. all the gear is ridiculously expensive and even kind of vain. It is not real camping. If you study the laws and rules or talk to rangers about the “right” way to camp, it would cost you thousands of dollars. That is not camping at all. Just like turning everything else into extreme money for the wealthy. The gadgets are not that complicated. Please do not purchase a 20 watt foldable solar panel for $199 that goes with the Goal Zero Yeti 400. I know you do not have that kind of money!
Jumper Cables: Sometimes for a couple different reasons, I found that my car battery died and I needed a jump. Most likely because I left the lights on or I charged my electronics too long without driving. It was a pain standing in front of a store asking people if they had jumper cables. I eventually got some jumper cables so when my car battery died, all I had to do was ask anyone who had a car around me if they could give me a jump rather than also having to ask them if they had jumper cables too.
Pet pads give you no choice. Human heat pads have low, medium and high. That means low and medium uses less wattage than high, which uses 50 watts. Pet pads require that you have a certain weight on them, so you cannot use them in any kind of situation. Human heat pads are the way to go, but make sure they do not have an automatic shut off. And a manual switch is better than digital, because you could accidentally turn off a digital and not realize it. The manual switch will sit where it is positioned regardless if you move things around, and in a vehicle things really do get in the way of other things.
You sure are right about people being judgmental and assuming we don’t work or pay taxes. When I tried to get a library card the librarian recognized my proof of address as the post office and said without a residential address I am not a citizen (I live, work and go to school in this city) and cannot have a library card because “The library is funded by the taxpayers.” I pay hefty self-employment taxes and taxes every time I buy something! Then, she went on to say I could have the non-resident card for $200/year. … I just went to another library in the system and they gave me a card. If you don’t pay property taxes you’re considered a second class citizen.
But there are other ways to cook healthy meals. When you’re camping, you can use the campfire to cook your meals. If you’re not at a campground, or it doesn’t have a fire pit, investing in a propane stove and cooking on the tailgate of your van will be a life saver, and will be a lot cheaper in the long run than eating out all the time. You can get a small camping stove for anywhere between $30-$150+.
I had a big scare a few nights ago. I parked in a business zone because I don’t like parking in a residential area so I won’t disturb the neighbors that could call the cops on me. Well, someone saw me and called the cops anyway. They came banging on my windows with flashlights. I sleep with a big black sheet on top of me so people won’t notice but since theywere looking in with flashlights they could see a body and even since I ignored them they continued banging with such a force I finally raised my head and they were shocked to see me and they asked me to open the door. Since it was hot, I was wearing little shorts so I climbed overn legs first, and I don’t mean to brag but I am fairly attractive and in great 24 hour fitness shape so they were a little flirty with me. They asked what I was doing, I said I was was resting. They asked if I had a home and I said yes. I had a fight with my boyfriend and I left and was just resting. I told them I didn’t know I was doing anything wrong. They asked if I was scared of gang activities. I said no, I wasn’t aware of this happening in the area. In my head I was thinking how I’m more scared of them because they could give me a ticket and impound my car and ruin my life even more. I apologized for doing something I didn’t know was wrong and told them I would go home now. I said good by and drove far away from that place and I will never go back there again.
But anyway, I have a dodge caravan with removable seats. Im short so I got all leg room i need. My problem is going to be heating and storage for my clothes and tools. I was thinking about using my friends portable propane grill for heat. Those little cans are fairly cheap. I have a power inverter for all my electronics up to 75w. The gym membership seems like a fantastic idea for hygiene.
During the recent economic climate, with uncertain employment prospects and staggering housing costs, some have even chosen to live in their cars as a strategic lifestyle. Pay off debt, simplify and de-clutter, avoid the yoke of an endless mortgage… Living in my car, proponents claim, means maximum flexibility with minimum overhead. In Walden on Wheels, a recent university graduate details his adventures in repaying $32,000 in student loans through taking odd jobs while having the adventures of a lifetime on the road — but he had an Econoline van, a palace compared with many modern sedans and compacts.
I’m a 63-year-old woman about to be homeless for the first time in my life. After living a hard-knock life (I’ll spare the details), I had a decent job with a decent wage, and was finally able to buy a little house, which I thought would be the beginning of a peaceful ease into retirement for me. Four months after I bought the house, I lost my job. I got unemployment for a while, but that ran out. I’ve been unable to find a decent job since then (it’s been 2 years now – I’m convinced that age discrimination is alive and well in the good ol’ USA), I was forced to start taking my Social Security as soon as I was eligible for it, because I had absolutely no other means of income. I also started the process of applying for a loan modification from the bank that has my mortgage (BofA), so that I could stay in my home, and continue to make my payments, but hopefully at a reduced interest rate, and lower payments that would be affordable on my SS income. After a year of endless hassle and fighting, not to mention tears and near mental breakdowns, I was denied an affordable loan modification, and I’m now facing a short sale. I’ve sold most of my furniture and all of my grandmother’s silver, as well as most of my own good jewelry, and I’ve rented a small storage unit for the few belongings that I feel I want to keep, and am now just waiting for “permission” to vacate my now-empty house. I do have a small SUV which is finally paid for, but I’m thinking about selling it and trying to buy a small RV to live in. My thought process is that I think I could afford moderate payments on a used RV, but I sure as heck know I can’t afford rent on a small apartment somewhere – unless anyone knows where I can get a decent (no ghetto or crack house, please) apartment for about $300 a month? My SS is only about $1,000 a month. Period. People I talk to think I’m crazy, but I seriously don’t see any alternative. No one knows how seriously depressing this is for me, or how close to suicide I have been over the past year. The only thing that keeps me going is my little dog, who I will never abandon.
Activation of the nuclear receptor and transcription factor CAR (Nr1i3) by its specific agonist ligand TCPOBOP (1, 4-bis[2-(3, 5-dichloropyridyloxy)]benzene) dysregulates hundreds of genes in mouse liver and is linked to male-biased hepatocarcinogenesis. To elucidate the genomic organization of CAR-induced gene responses, we investigated the distribution of TCPOBOP-responsive RefSeq coding and long noncoding RNA (lncRNA) genes across the megabase-scale topologically associating domains (TADs) that segment the genome, and which provide a structural framework that functionally constrains enhancer-promoter interactions. We show that a subset of TCPOBOP-responsive genes cluster within TADs, and that TCPOBOP-induced genes and TCPOBOP-repressed genes are often found in different TADs. Further, using DNase-seq and DNase hypersensitivity site (DHS) analysis, we identified several thousand genomic regions (ΔDHS) where short-term exposure to TCPOBOP induces localized changes (increases or decreases) in mouse liver chromatin accessibility, many of which cluster in TADs together with TCPOBOP-responsive genes. Sites of chromatin opening were highly enriched nearby genes induced by TCPOBOP and chromatin closing was highly enriched nearby genes repressed by TCPOBOP, consistent with TCPOBOP-responsive ΔDHS serving as enhancers and promoters that positively regulate CAR-responsive genes. Gene expression changes lagged behind chromatin opening or closing for a subset of TCPOBOP-responsive ΔDHS. ΔDHS that were specifically responsive to TCPOBOP in male liver were significantly enriched for genomic regions with a basal male bias in chromatin accessibility; however, the male-biased response of hepatocellular carcinoma-related genes to TCPOBOP was not associated with a correspondingly male-biased ΔDHS response. These studies elucidate the genome-wide organization of CAR-responsive genes and of the thousands of associated genomic sites where TCPOBOP exposure induces both rapid and persistent changes in chromatin accessibility.
Most small vehicle dwellers rely on a combination of plastic boxes and duffel bags. Boxes or bins are hard-sided and can offer protection to items that might be crushed or damaged, but take up the same amount of space even when empty. Duffel bags do not offer much protection for the contents, but do collapse to take up less space when they are not full.
If you are in a situation where you have some warning that your financial situation is going to lead to you having to live in a car, or that your house is no longer going to be your home, then plan ahead. If you are struggling to make loan repayments despite a reasonable income and talking to the bank has failed, then perhaps it is better spending those last repayments on buying a van instead of paying off a hopeless loan? Even if you are laid off, is there anything you can sell that will get you enough money to buy a van or camper? If you are in a rocky relationship and about to face a divorce, can you buy a campervan / RV before it all goes sour?
In your first few nights, you will likely feel very vulnerable and even uncomfortably furtive, with an unaccustomed sense of needing to “lay low” and keep out of view of the authorities. Luckily, there are some reliably hassle-free places to spend the night, provided you practice good-neighbor leave-no-trace habits. Drive to your camping spot when you are ready for sleep (having completed your nightly hygiene routine), cut the engine and settle in as quickly as possible, avoiding noise and excess lights. For best results, rotate your spots so that nearby residents don’t start noticing you and making complaints. Use common sense and local advice to steer clear of high-crime neighborhoods, as well as isolated urban areas where you will be more vulnerable. Having others around, in general, is good; though your earplugs will come in handy to block out voices, door-slamming, and loud footsteps.
Due to child support from three seperate states for three kids I have been living out of cars and in tents for eight years now. Being homeless is alot easier when you get farther away from mainstream society and camp. If you can afford it stay in a campground with showers etc. If not try finding a quiet place on the river or lake somewhere. Or up in the hills close to a spring or small stream. I hated being homeless in the city but popping a tent in the wilderness is not that bad and is good for the soul.Nature will provide all that a person needs if you know where to look and how to use what is available. Circumstances are different depending on the region you are in. Personally I have a strong spiritual connection to the forests that I frequent and over time a person will develop this. The more you realize that you are a part of the earth the more in tune you become with your surroundings. You do not own the earth. The earth owns you. lol.
I bought a cheap gym membership close to the center of the city. I store a couple towels and my gym clothes in a locker and lock it so I dont have to store it in my car. The gym has signs saying you have to pay to rent a locker, if you want to keep your stuff there overnight, and all lockers not paid for can be cut and stuff removed by staff. I just change lockers every couple days or every day if I feel paranoid, Its a cheap gym how are they going to know.
Truck life, on the other hand, facilitates simplicity. “When you don’t own many things, you don’t worry about many things,” Cochrane says. “I’m the happiest I’ve been in years, and this is surely part of that function.” That happiness also probably stems from his now-enhanced ability to run, ski, and adventure more freely, as well as greater flexibility to spend more time visiting friends, meeting new people, and taking more ocean swims and fewer actual showers.
You’ll also need to know where local public washrooms are, especially if you’re a woman. In areas where using a store’s washroom without buying something is frowned upon, use the washrooms of large stores, like a grocery store, where you won’t be noticed. If you’re in the woods, or male, you may also find use for a child’s training toilet and a few rolls of toilet paper come in handy
I live in Florida. I’m 49, married and have two kids who are about to start college. My wife and I met in college and have been working in corporate America since we were teenagers. We are doing very well and life has been simple but good to us. However, I’m terribly overweight and suffering from anxiety and general lack of interest. Physiologically, I’m getting near my breaking point. Lately, all I do is dream about running away from everyone and everything. Even though our net worth is very high, I dream of buying a VW Budd-e EV Van and living out of it with just a cell phone, laptop and 3 pairs of jeans. I imagine I could shower in 24 hour fitness and eat out every day. I could just drive up and down the east coast, parking at the beach or charging stations near points of interest. Just to test the theory, I’ve take short hall-pass trips for 2 – 3 day and done it. It felt amazing and liberating to not to have a schedule to be that free. I never felt unsafe or uncertain like I do in my regular life.
And they NEVER once tell me who did the accusation, who did the lie, the attack, after all those months. “We deframe your character and tell it to your face all of a sudden and there is nothing you can do about it.” Like a case and jury and trial you heard nothing about is all shut over and done. Three times i just blasted them right back in the face all of a sudden, I mean with a loud strong voice, right into their face, so that the entire block or business would turn around and stare or hear clearly – because they do those things intimidation, defamation, and lies in silence or hidden. They don’t even come with proff because they think poor people are easy to shove around. But and only one time did I receive the rightful apology from the boss over the lies and abuse, so that I could continue to use what I was using before.
Overtime, I was able to recover. I was also fortunate enough to have three years in remission with normal-range liver enzyme results until this past October 2017, when I relapsed with my AIH. I am managing with the relapse and I am thankful and lucky to have had the three years of remission. I know this is not indicative of most people with AIH. It gave my body the chance to recover that many people living with AIH don’t get the opportunity to experience.
I am male 38 years old and I have lived in my 2005 Prius in Dallas,Texas for 9 months. I have chosen to adapt my life to living out of a car to see what life it might create. After getting rid of 85% of my stuff and buying a Dreamtime Therma-rest pad from REI and a piece of plywood to extend the backseat by 4 inches beyond the seats laying down, I am living in plushville. So far missing a internet connection in the car has been the biggest thing i would like. I am thinking the 3G iPad will be my next logical leap. I also realized that past 85 degrees sleeping in my car is unpleasant ( i am a bit of a princess and i prefer not to sleep with the car on except in extreme weather conditions). I need to procure a solar powered fan with a battery by next summer (note to self).
Unless your car is knocking and pinging, don't use a higher octane gas than your owner's manual recommends. For most cars, premium gasoline offers no benefit. Unless your car has a high-performance engine and your manufacturer recommends a high-octane gas, use the less expensive gas. Premium gas costs 10% to 15% higher than regular. Potential Money Savings: $200-400/yr.
im gonna be living out of my mini-van for awhile. just graduated college and dont feel like going into the work world right away. just wanna drive around, travel, explore. i hate running into cops. For awhile I didn’t have internet in my house, so I would drive around to the nearest public place to surf the web. This led me to have so many run ins with cops. hate it. i can’t imagine how hard it’ll be to avoid cops when i’m living out of your van. I’m planning on going to the southern tip of florida in order to be in the warmest place possible for the winter. I wonder if it will still get too cold there at night during january?
If you can't afford a porta potti there are other options. A basic bucket half filled with water and detergent will serve as a potti. It will smell though. A layer of oil on top of the water will help keep the smell down. But that can be messy. You can use a bucket filled with sand, but that gets smelly too. A bucket filled with kitty litter works pretty good. You can pee into bottles, Gatorade bottles do well.
When you park, think about which direction the sun is coming from. In summer, you may want to seek out a shaded spot to help keep cool. In winter, you might want to seek a sunny spot, and face the car into the sun to allow as much heat in through the windscreen. Generally though, you should face the car so that you can drive forward out of the place you are parked in. In an emergency, you don't want to have to back up to leave. Some people say it is best to leave the keys in the ignition while others say it is best not to. Me, I have found I have felt safer with the keys in my pocket, or close to the ignition where people can not see it. When I slept away from towns I did not always block out the windows, so the keys would be out of the ignition. When I sleep with privacy curtains I leave the keys in the ignition.
I live very frugal cutting expenses at every corner in a series of routines here is how I do it. I live in a smaller city (about 300,000 people) in California. The car I bought is a smaller car (I’m a small dude) with large trunk which is perfect for me, it gets great fuel mileage, even though its tight, it has enough space to store my stuff and still be livable. . I keep it spotless inside and out, dark purple, looks like a granny car. I keep all my stuff extremely neatly concealed at all times. The back of the rear seats fold down over the top of the back seat to expand storage. As far as general public and cops go the car is invisible.
I recently upgraded to a better parking lot at the, Hyatt Place Hotel parking lot. I joined a gym for $9.00 per month with no contract. Therefore, I work in the Virginia, D.C., Maryland job market as an Integration Supervisor, but I rent a nice house for $800.00 per month in eastern North Carolina by the water and go down there every two weeks and sleep in my bed and enjoy the water, marinas and sunrises and sunsets. I have lived in my car for about a year, in the freezing cold of winter and hotest day of summer. But being able to adapt and being thankful for every day I am alive is the key. All the best to those who challenge the status quo and live to inspire others to do what inspires them.
Find a local laundromat! Some have wifi so you can get work done while you do your laundry. You can also buy detergent and do laundry in whatever bathroom sink you can find, but you’ll have a tougher time getting clothes dry quickly. My biggest laundry problem was traveling with a big fluffy cotton towel. The towel would take forever to dry after a shower and would always start to smell funny. The camp towels you can find at your outdoor goods stores were less bulky and dried faster, but would never seem to dry as well as a cotton towel.
I was a security officer for about 13 years, and I can truly tell you what cops and security look for: Things that are out of place…things that don’t seem like they belong there. For example, in an apartment complex, if you park with the other vehicles (blend in), and keep a low profile (no lights at night…cigarettes, lighters, reading lights, etc), then you probably won’t get noticed. However, if you park in a secluded spot, I can almost guarantee that you will get the attention that you don’t want. Cops and (professional, trained) security look at things in black and white: Normal, or NOT NORMAL. It’s the “NOT NORMAL” things that trigger a response. Keep that in mind and you’ll do fine. 🙂
Speaking of the rich; some are okay people. But there are rich people—-one I know inparticular who inherited money from Rolls Royce who does nothing but complain. He’s gay too, and always calls the guys down at my desk to come up to his penthouse to take care of noises and BS just to get them in his place. He made a pass at me a few times. People like this have no values in life. And from one trip to Aruba to the Virgin Islands and back to their cribbs, their only problems along the way may be a loud noise on the roof, a spot on the pillow, or coffee not hot enough.
Get some cashmere sweaters, second hand stores don’t even know what it is, 3 bucks for cashmere. and it is washable by hand. but the new fabrics for skiers are great too. A wool hat and some goose down comforters. I find that having a hat on at night and wool socks helps a lot. Goose down is vital wool blankets too. I put them into bags during the day so the dog stays off of them. He slept in the front seat. My cat had a pet box and went outside during the day. I parked at a place where she could go out and find me at night. (small fourplex with a large parking lot). My horse was bored and I had a tack room where I stored my things.It is easier to bored a horse in the city, then find an apartment.
I am in Europe (English person in Luxembourg) and have just read the above posts. Some excellent info there, thanks. I am 14 days away from the same situation except the car is going too. I am considering buying a 500 euro (650US) Ford Fiesta type car, maybe something a little bigger as Renault Megane type cars have a low resale. But insurance etc will be expensive and require an address…. I can earn some money as an English (as a foreign language) teacher but need to look the part; especially for the adult students who tend to work in banks and therefore have higher standards. I also want to keep fit and so will continue to swim and shower by the pool (before entering water and after!). I think shaving and teeth cleaning (English people do you know) will not be accepted by other users so that will be a no-no. This is not like America so a parked car not in a residential street will attract attention, especially if old and cheap. There are homeless people here and a community that drinks beer in the square but I prefer to avoid that (I don’t know where they sleep, altho they are well dressed and so i guess they get clothes from some charity). I have read about US tent cities and that would be great here too but not likely to happenas lack of the outdoors would be a problem (everything/where is private). So my plan B is to live in a small survival tent. This can be carried in a pack (I hope) and if I walk off the track in the woods each evening and put up the tent in a densely treed or ferned area, i should be almost invisible including from above. I am worried that I am living in a fantasy and will freeze to death. In winter, it can be -15 C (5F). This site gives me hope. I hope that all or most of the posts are real.. Anyway, I have been reading about little one man tents and that seems fine. The sleeping bags seem Ok too. I have around 800 dollars in cash and can earn enough to eat / drink / swim but nothing else. I have clothes / books etc and I am not sure what to do with them. Storage sounds good but too expensive (unless i live in it but that seems too difficult to get away with). So nearly everything will have to go although some can go with friends (who i am not telling about this). But I can buy a few things to survive/live. I am 54 and in reasonable shape at 160 pounds. My first question is whether it is feasible to sleep in one of those blizzard survival bags. Are they just a one day thing or could they perhaps go inside or outside my bag? Don’t tell my ex but the thing I will miss most is my two cats. I dream about keeping them in a tent to keep me company and keep my feet warm but sadly that IS a fantasy. So blizzard bags? Anybody tried living without the car or even keep the car, possibly to store some stuff in the boot (trunk) but sleep outside it to avoid police attention. Sorry for the long post but I have little time before I leave the nice flat that I am currently sitting in. Thanks, tim
I still have to pay for gas and so forth, but I am “out there” all day. I don’t feel lonely as I would in my house and I love not having to pay heating for the whole condo. I had a problem with knots in my shoulder from working on the internet bent over in my car but I found if go to the gym and use the exercise equipment for my shoulders and pull on weights and life stuff then knots are not a problem anymore.
Our suggestion? Get a cargo rack. These are great to get a fair bit of your gear out of the car and onto the roof. Most, if not all, boast a secure lock so theft isn’t an issue, and all that newfound free space will not only prevent you from looking like a destitute hoarder but will open up the cabin for your sleeping arrangements. A win, win if you ask us. It’s always been the risk-takers, go-getters, and – for lack of a better word – weirdos who change the world and live lives worth living. Also, something as simple as a couple storage bins could go a long way as well to keep things organized and tidy when traveling/living out of your car. Again, if you have the time and money, a custom build-out is always the way to go, and there are plenty of aftermarket workshops that can do this for you based on any sort of van-life inspiration you may have come across.
Using an air freshener in a car that smells awful is a lot like pushing all your dirty clothes under the bed when your mom tells you to clean your room. However, they’re better than nothing. Your vehicle is going to smell bad no matter what you do, especially if you’re extremely active and go hiking, biking, running etc. all the time and get sweaty and gross, but using air fresheners and replacing them often will make your nose at least a little bit happier, and every bit helps. Our personal favorite scents were black ice and forest pine, for the record.
CAR activation induces β-oxidation in the liver. (A) Ob/ob and ob/ob CAR−/− mice were treated with TC or corn oil control for 1 month. Liver samples were used for measurement of a series of acylcarnitines with fatty acyl side chains of the indicated lengths, and also the indicated organic acids by stable isotope dilution mass spectrometry. (n = 5, *P < 0.01) (B) Ob/ob and ob/ob CAR−/− mice were treated with TC or corn oil control for 1 month. Mice were fasted for 6 h and serum ketone bodies were measured. (n = 6, *P < 0.05) WT and CAR−/− mice were treated with TC or control for 3 days. Primary hepatocytes were measured and performed β-oxidation assay. (n = 3, *P < 0.01) (C) Liver total RNA from 1 month treated ob/ob and ob/ob CAR−/− mice were examined for ACC1 and ACC2 expression. (n = 4, *P < 0.01)