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Losing Weight as a Couple Is the Secret to Shedding Pounds, Study Finds – Best Life
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On the list of things that are easy to pull off, losing weight might be at the very bottom for most people. While setting up a diet plan and making a commitment to hit the gym always feels good on day one, busy schedules and loss of enthusiasm can take a toll on achieving your goal. But a new study says there's one secret trick to shedding pounds that you probably haven't tried that is proven to show results: losing weight with your spouse.
The proof comes from a study conducted by the European Society of Cardiology, which assessed 411 heart attack survivors attempting to make some lifestyle improvements in areas such as quitting smoking, increasing exercise, and losing weight. The data showed that participants who pledged to make changes with their spouse saw a much greater success ratethey were more than twice as likely to improve in at least one of the areas. But results also showed that of the three categories, being a part of a pair made weight loss much more successful.
"Patients with partners who joined the weight loss program lost more weight compared to patients with a partner who did not join the program," study author Lotte Verweij, a registered nurse and PhD student at Amsterdam University of Applied Sciences in the Netherlands, said in a statement.
Verweij also pointed out that underlying factors may be the secret to this tactic being so effective. "Couples often have comparable lifestyles and changing habits is difficult when only one person is making the effort," she said. "Practical issues come into play, such as grocery shopping, but also psychological challenges, where a supportive partner may help maintain motivation."
Getting in shape with your spouse tends to work better than going it alone because of increased accountability, immediate support, and shared workloads on things like meal prep. But setting expectations ahead of time can also help avoid any unnecessary stress between the two of you.
"Don't expect the people in your life to be mind readers," Patrick O'Neil, PhD, director of the Medical University of South Carolina Weight Management Center, told My Fitness Pal. "Let [your spouse] know that you wish them to be helpful and how you'd like them to support and help you."
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Still looking for ways to lose weight as a couple? The experts at Shape suggest things like setting up a meal calendar at the beginning of each week, getting outdoors, romantically splitting a single dessert, or taking a dance class together to get started. And for more on how to take care of yourself in the long run, avoid these 45 Unhealthy Weight Loss Tips Experts Say to Avoid at All Costs.
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Losing Weight as a Couple Is the Secret to Shedding Pounds, Study Finds - Best Life
Looking to lose weight? Try these new recipes from Scottish Slimmers’ new cookbook – The Courier
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Give yourself a nutritional boost and shed the pounds with these two plant-based recipes to get you started on your journey to a healthier you
With gyms having been closed and the fridge a lot closer to us than usual these past few months, very few of us have managed to emerge from lockdown without a bit of extra weight.
But, there is some good news. Weight-loss experts Scottish Slimmers are about to launch a new cookbook, filled to the brim with healthy vegetarian recipes designed to help you lose the weight while still eating your favourite foods completely guilt-free.
We previously showcased Scottish Slimmers recipes for a black bean burger and a sweet potato and chickpea curry, but this week we have another two equally-delicious recipes to share, including a stir-fry and a healthier take on spaghetti and meatballs.
(Serves 2)
Fat: 7.7 gCarbs: 60 gProtein: 10 gTotal Calories: 348
(Serves 2)
Fat: 14gCarbs: 43 gProtein: 13gTotal Calories: 335
As study shows Scots ate more healthily in lockdown, the slimming recipes to help you keep going
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Looking to lose weight? Try these new recipes from Scottish Slimmers' new cookbook - The Courier
Jennifer Aniston was asked to lose weight if she wanted to stay on Friends – The News International
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Hollywoods leading lady Jennifer Aniston has been reigning over the industry for decades now, courtesy of her iconic role on the sitcom, Friends.
While the show catapulted her to great heights of fame, the role almost slipped away from Anistons hands owing to the makers insistence on her losing weight.
The revelation was made by Saul Austerlitzs book Generation Friends: An Inside Look at the Show that Defined a Television Era.
She had to lose thirty pounds if she wanted to stay in Hollywood. Los Angeles was a tough place to be an actress it was a tough place to be a woman and Jennifer Aniston's agent was reluctantly leveling with her, wrote Austerlitz.
With her father already being a soap opera star, Aniston was familiar with how tough the industry can get.
"Aniston was hardly fat everyone could see she was beautiful but as the show she would one day become indelibly associated with later made a point of noting, the camera added ten pounds," the author claimed.
During an earlier interview in 1996 with the Rolling Stone, Aniston too had opened up about her struggle with weight loss: "I ate too many mayonnaise sandwiches. Mayonnaise on white bread the most delicious thing in the world.
"My agent gave it to me straight. Nicest thing he ever did. The disgusting thing of Hollywood I wasn't getting lots of jobs 'cause I was too heavy. I was like, 'What?!' But my diet was terrible. Milk shakes and French fries with gravy, she recalled.
"It was a good thing to start paying attention, she added.
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Jennifer Aniston was asked to lose weight if she wanted to stay on Friends - The News International
Global Weight Loss & Diet Management Market 2020 | Scope of Current and Future Industry 2025 – Scientect
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This report focuses on the Global Weight Loss & Diet Management Market trends, future forecasts, growth opportunities, key end-user industries, and market players. The objectives of the study are to present the key developments of the market across the globe.
The latest research report on Weight Loss & Diet Management market encompasses a detailed compilation of this industry, and a creditable overview of its segmentation. In short, the study incorporates a generic overview of the Weight Loss & Diet Management market based on its current status and market size, in terms of volume and returns. The study also comprises a summary of important data considering the geographical terrain of the industry as well as the industry players that seem to have achieved a powerful status across the Weight Loss & Diet Management market.
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Weight Loss & Diet Management Market Segmentation
By Diet Meal, Beverages, Supplements, OtherBy Equipment Fitness Equipment, Surgical Equipment, OtherBy Service Fitness Centres, Slimming Centres, Consulting Services, Online Weight Loss Programs, OtherBy Distribution Channel Multi-level Marketing, Large retail, Small Retail, Health & Beauty Stores, Online Distribution, Other
The report has been curated after observing and studying various factors that determine regional growth such as economic, environmental, social, technological, and political status of the particular region. Analysts have studied the data of revenue, production, and manufacturers of each region. This section analyses region-wise revenue and volume for the forecast period of 2015 to 2025. These analyses will help the reader to understand the potential worth of investment in a particular region.
Global Weight Loss & Diet Management Market: Competitive LandscapeThis section of the report identifies various key manufacturers of the market. It helps the reader understand the strategies and collaborations that players are focusing on combat competition in the market. The comprehensive report provides a significant microscopic look at the market. The reader can identify the footprints of the manufacturers by knowing about the global revenue of manufacturers, the global price of manufacturers, and production by manufacturers during the forecast period of 2015 to 2020.
The major players in the market GlaxoSmithKline, Herbalife, Abbott Nutrition, Nestle SA, Danone, Glanbia, , Pepsico, Atkins Nutritionals, NutriSystem Inc., Jenny Craig Inc., Creative Bioscience, Iovate Health Sciences, Nutrisystem, Ethicon, Apollo Endosurgery, Brunswick, Amer Sports, Johnson Health Technology, Technogym, Weight Watchers, VLCC Healthcare, Slimming World, The Golds Gym International, Duke Diet & Fitness Center, Kellogg Company, Medifast, Inc., Kraft Foods Inc., General Mills Incorporation, Amylin Pharmaceuticals, lpro Ltd., Ajinomoto Co. Inc., AIDP Inc., AHD International, Acatris Inc., Zydus Cadilla Healthcare, Health Biotech Ltd., Olympus Corporation, 24 hours Fitness, Fitness First Group, Town Sports International Holdings Inc., Pfizer, Stepan, American Health, FANCL, Natures Sunshine Products, Amway (Nutrilite) and others.
Global Weight Loss & Diet Management MarketThis research report providesCOVID-19 Outbreakstudy accumulated to offer Latest insights about acute features of the Weight Loss & Diet Management Market. The report contains different market predictions related to marketsize, revenue, production, CAGR, Consumption, gross margin, price, and other substantial factors. While emphasizing the key driving and restraining forces for this market, the report also offers a complete study of the future trends and developments of the market. It also examines the role of the leading market players involved in the industry including their corporate overview, financial summary andSWOT analysis.It presents the360-degreeoverview of the competitive landscape of the industries. Weight Loss & Diet Management Market is showing steadygrowthandCAGRis expected to improve during the forecast period.
The main sources are industry experts from the global Weight Loss & Diet Management industry, including management organizations, processing organizations, and analytical services providers that address the value chain of industry organizations. We interviewed all major sources to collect and certify qualitative and quantitative information and to determine future prospects. The qualities of this study in the industry experts industry, such as CEO, vice president, marketing director, technology and innovation director, founder and key executives of key core companies and institutions in major biomass waste containers around the world in the extensive primary research conducted for this study We interviewed to acquire and verify both sides and quantitative aspects.
Global Weight Loss & Diet Management Market: Regional AnalysisThe report offers in-depth assessment of the growth and other aspects of the Weight Loss & Diet Management market in important regions, including the U.S., Canada, Germany, France, U.K., Italy, Russia, China, Japan, South Korea, Taiwan, Southeast Asia, Mexico, and Brazil, etc. Key regions covered in the report are North America, Europe, Asia-Pacific and Latin America.
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Complete Analysis of the Weight Loss & Diet Management Market:
Comprehensive assessable analysis of the industry is provided for the period of 2020-2025 to help investors to capitalize on the essential market opportunities.
The key findings and recommendations highlight vital progressive industry trends in the global Weight Loss & Diet Management market, thereby allowing players to improve effective long term policies
A complete analysis of the factors that drive market evolution is provided in the report.
To analyze opportunities in the market for stakeholders by categorizing the high-growth segments of the market
The numerous opportunities in the Weight Loss & Diet Management market are also given.
Report Answers Following Questions:
What are the factors driving the growth of the market?
What factors are inhibiting market growth?
What are the future opportunities in the market?
Which are the most dynamic companies and what are their recent developments within the Weight Loss & Diet Management Market?
What key developments can be expected in the coming years?
What are the key trends observed in the market?
TABLE OF CONTENT
1 Report Overview
2 Global Growth Trends
3 Market Share by Key Players
4 Breakdown Data by Type and Application
5 United States
6 Europe
7 China
8 Japan
9 Southeast Asia
10 India
11 Central & South America
12 International Players Profiles
13 Market Forecast 2020-2025
14 Analysts Viewpoints/Conclusions
15 Appendix
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Global Weight Loss & Diet Management Market 2020 | Scope of Current and Future Industry 2025 - Scientect
Can the Pioppi Diet really cut your Covid risk? – Spectator.co.uk
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A recent book published by Dr. Aseem Malhotra, already on the Sunday Times bestseller list, claims that within 21 days, people with obesity or who fall on the spectrum of metabolic disease, can reduce their susceptibility to SARS-COV2 infection severity by following this simple formula: eat real food, move, relax, sleep.
A rather bold claim to make, considering that the UK death toll from SARS-COV2 infection currently stands at around 41,500. A provocative, editor-selected headline that was subsequently replacedsuggested that this was the best vaccine, and predictably produced a spirited, nuanced and reasoned reaction on Twitter.
The question one has to ask is: is there any evidence, for this claim? Is the Malhotra method the solution, or are his more strident critics justified in their passionate skepticism?
Multiple studies of different designs have addressed the impact of metabolic disease, insulin-resistance, diabetes, hypertension, obesity, sleep deprivation, stress and exercise on overall health, immunity and the ability to withstand a multisystem infection and it is to these that one must look in order to assess the potential veracity of Malhotras claims. We have recent studies that have established that all of these disease states increase the susceptibility of patients, to complications of SARS-COV2 infection.
Morbid obesity is associated with a 50 per cent increased risk of dying from SARS-COV2 infection and doublesthe risk of hospitalization. Once hospitalized, obese patients are more likely suffer complications should intubation be required.
Is there an easy method whereby someone can begin to address their obesity given the current restricted access to non-SARS-COV2 NHS services in many areas? Indeed there is. We know that consumption of ultra-processed foods (UPFs) cause excess calorie intakeby not being satiating, and that they directly contribute to obesity and the development of insulin resistance and diabetes.We also know that 60 per cent of the total caloric intake in the UK consists of UPFs. By avoiding UPFs, one automatically reduces total caloric intake.
Metabolic disease exists along a spectrum. There are five criteria that establish abnormal values in regard to waist circumference, triglyceride and HDL levels, blood pressure and fasting blood glucose values. Anyone possessing one of the criteria is on that spectrum. A patient with three or more of the five criteria is diagnosed with metabolic syndrome.
Not everyone who is obese will have metabolic syndrome and not everyone with metabolic syndrome is obese. 88 per cent of Americans and possibly the same proportion of people in the UK, are metabolically unhealthy regardless of their weight.
Insulin resistance is a fundamental part of the metabolic disease spectrum and has a deleterious effect on the immune system, partly explaining why those with it are at higher risk of severe SARS-COV2 infection.
Angiotensin Converting Enzyme 2 (ACE2) receptors serve as an attachment point for the virus as it enters human cells; receptors for this enzyme are found in the lungs, among other tissues. Elevated blood levels of insulin and glucose, lead to higher levels of this enzyme and its receptors. They also contribute to the cytokine storm which results from an abnormal immune response which causes multi-organ damage in severe SARS-COV2 infectionas well as impaired activity of several important cells of the innate immune system.This propensity for infection has been demonstrated with another corona virus, H1N1.
The observable impact of metabolic syndrome on SARS-COV2 infection severity is profound. A three-fold greater risk of death is noted,as is a five-fold higher risk of needing intensive care or a ventilator; similar risks are seen for diabetes and hypertension.
Higher blood sugar levels alone, in the non-diabetic range, are also a risk factor for death within 28 days of hospital admission.
So can metabolic disease (insulin resistance) be improved, treated or cured via non-medication manipulation?
A program of stress reduction, has been associated with improved insulin resistance given that stress itself can cause insulin resistance.Sleep deprivation can rapidly cause insulin resistance. But interestingly, high-intensity exercise can mitigate this effect.
Exercise of any kind, itself, has been shown in multiple studies to improve insulin resistance, independent of any weight loss.
A meta-analysis of randomized controlled trials, supports a low-carbohydrate diet for patients with type 2 diabetes and metabolic diseasewith improvements in insulin level, blood sugar and inflammatory markers demonstrable.
A smaller observational study suggests that rapid improvement in markers of insulin resistance and cardiovascular risk factors may be seen in as little as two weeks with carbohydrate restriction.
Lest anyone think I am pushing the low-carb agenda, another study found that a low-fat, high-fibre diet in conjunction with exercise was associated with significant biochemical improvement; 50 per cent of patients experienced reversal of metabolic syndrome within 21 days.
For those people with obesity and metabolic syndrome, modest weight loss was associated with significant improvements in markers of metabolic syndrome or even resolution within a 4 week period.
Improvements in biochemical markers for metabolic syndrome can be seen within 9 days simply by substituting processed sugars for starch, without altering the caloric content of the rest of the diet.
Clearly there is some merit to what Malhotra is recommending. Given that metabolic disease, diabetes and cardiovascular disease are among the most significant risk factors for severe SARS-COV2 infection, ameliorating their impact on the health of vulnerable people should be a non sequitur.
One wonders why those with the power to affect change have not been taking advantage of the opportunity lockdown protocols have afforded to improve the nations health via a sustained focus on metabolic health improvement strategies. Given that metabolic disease and obesity disproportionately affect the poorer members of society, there should be all the more impetus to intervene.
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Can the Pioppi Diet really cut your Covid risk? - Spectator.co.uk
Watch What Happened When This Guy Ate and Trained Like NBA Legend LeBron James for a Day – menshealth.com
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Following his videos where he followed the diets of actors like Hugh Jackman, Tom Ellis and Arnold Schwarzenegger, YouTuber Aseel Soueid felt like it was time to take on the GOAT. In his latest video, he spends the day living like basketball legend LeBron James; that means taking on the NBA star's grueling workout, and then eating his favorite meal.
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Soueid kicks off the workout with LeBron's favored weight training combo, starting with 4 sets of 12 on the deadlift. "I'm already remembering why I don't do deadlifts," he says after his first set. "They kick your ass... It really really engages the core, legs, hamstrings, back, the whole nine yards."
He follows this with 4 sets of 12 reps on the standing barbell curl, and 4 sets of 12 on the barbell bent over row.
Then it's time for the bodyweight exercise portion: 3 sets of 20 wide-grip pushups, which he completes with relative ease, and 3 sets of 15 chinups. "Mad respect to LeBron James," he says. "3 sets of 15 is a lot of chinups. For a guy that size, chinups are a lot harder than you think." He adds that usually this number of reps would be "effortless" for him, however, coming right off the deadlifts and barbell rows, he's struggling with his grip strength, and he has to take 2 to 3 minutes to rest between each set.
Soueid rounds off the LeBron workout with 30 minutes of yoga, which helps with mobility when it comes to all of the other intensive training.
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And then, finally, it is time for the post-workout meal, which in this case, is a recreation of LeBron's exact Blaze Pizza order, inspired by his stake in the restaurant chain. The pizza is topped with spicy red sauce, mozzarella, parmesan, grilled chicken, turkey meatballs, banana peppers, cherry tomatoes, garlic, basil, green peppers, olives, red onion, spinach, sea salt, arugula, and olive oil. This is accompanied by an entree-sized salad with chicken breast.
Soueid's verdict is simple. "I'm so freaking happy right now," he says. "It's worth all those 4 sets of 12."
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Watch What Happened When This Guy Ate and Trained Like NBA Legend LeBron James for a Day - menshealth.com
Which cooking oil is the healthiest? – BBC News
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Cooking oils are a kitchen staple. But theres a lot of conflicting information regarding how healthy each of them are. With so many on the shelves from coconut to olive, vegetable to canola, avocado to rapeseed oil how do we know which ones to use, and if we should be avoiding any altogether?
Oils used for cooking tend to get their name from the nut, seeds, fruits, plants or cereals theyre extracted from, either by methods of crushing, pressing, or processing. Theyre characterised by their high fat content, including saturated fat, monounsaturated and polyunsaturated fatty acids.
In recent years, coconut oil, which is around 90% saturated fat, has become the latest trendy superfood. Its been hailed as a superfood (including that it's less likely to be stored in the body as fat and more likely to be expended as energy) but one Harvard University epidemiologist calls it pure poison.
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Consuming too much saturated fat more than 20g for women and 30g for men per day, according to UK guidelines makes the body produce cholesterol in our bodies that increases the risk of heart disease.
All fat molecules are made of chains of fatty acids, which are either held together with single bonds (saturated) or double bonds (unsaturated). There are three types of fatty acids: short, medium and long chain. Short and medium chain fatty acids are absorbed directly into the bloodstream and used for energy, but long chain fatty acids are transported to the liver, which raises blood cholesterol levels.
Coconut oil enjoyed popularity three or four years ago, when there were claims it had a special effect, says AliceLichtenstein, Gershoff professor of nutrition science and policy at Tufts University in Massachusetts, US.
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Which cooking oil is the healthiest? - BBC News
Suffering from excessive hair loss? Try these expert diet tips to reduce hair fall and breakage – Times Now
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Suffering from excessive hair loss? Try these expert diet tips to reduce hair fall and breakage | Photo Credit: iStock Images
New Delhi: Hair is the part of the body that enhances beauty. Nowadays, Hair Fall is a major problem that is being faced by many people, especially during monsoon. But Why? This is because of the lifestyle that we follow. Pollution, dust, improper diet, use of chemical products etc results in dry hair fall. Direct exposure of hair to the sun also makes your hair dry and weak.Also, people generally face dryness of tresses and scalp. For stopping rainfall, people visit doctors for transplant, medicine but we can prevent hair fall or reduce naturally by our meals. Eating healthy food reflects the health of hair.
Diet is good for hair and prevents hair fall. Also, it provides growth and volume to the tresses.
Nutrients like vitamin A, C, D, E, protein, zinc, iron etc are healthy for hair. These nutrients make hair strong, shiny and promote growth. Many people apply ingredients like egg, yoghurt, aloe vera etc on their scalp and tresses, this is good to some extent. But, if you don't have healthy meals(nutrients), applying things won't work on the health of hair.Pooja Banga, Director and Nutritionist at Cultivating health, suggests some foodsthat one should include in their diet for healthy hair:
To prevent hair fall or dryness of hair, you should eat these food items. Lack of nutrients like vitamins, zinc, protein, iron etc, makes hair dull and weak. Try to avoid junk food as it is not healthy for hair and skin as well. A healthy diet is really important for strong, shiny and voluminous hair. If you think you are lacking these nutrients, start taking them and feel the change in your hair strength. Take care of your hair and stay beautiful!
Disclaimer: Tips and suggestions mentioned in the article are for general information purpose only and should not be construed as professional medical advice. Always consult your doctor or a dietician before starting any fitness programme or making any changes to your diet.
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Suffering from excessive hair loss? Try these expert diet tips to reduce hair fall and breakage - Times Now
Ancient Beringian paleodiets revealed through multiproxy stable isotope analyses – Science Advances
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Abstract
The earliest Native Americans have often been portrayed as either megafaunal specialists or generalist foragers, but this debate cannot be resolved by studying the faunal record alone. Stable isotope analysis directly reveals the foods consumed by individuals. We present multi-tissue isotope analyses of two Ancient Beringian infants from the Upward Sun River site (USR), Alaska (~11,500 years ago). Models of fetal bone turnover combined with seasonally-sensitive taxa show that the carbon and nitrogen isotope composition of USR infant bone collagen reflects maternal diets over the summer. Using comparative faunal isotope data, we demonstrate that although terrestrial sources dominated maternal diets, salmon was also important, supported by carbon isotope analysis of essential amino acids and bone bioapatite. Tooth enamel samples indicate increased salmon use between spring and summer. Our results do not support either strictly megafaunal specialists or generalized foragers but indicate that Ancient Beringian diets were complex and seasonally structured.
Identifying the subsistence strategies of the earliest inhabitants of the Americas remains a contentious problem; these populations have been portrayed as megafaunal specialists or as broad-spectrum foragers (1, 2). Addressing this issue is a key in understanding the initial colonization, routes of dispersal, and settlement of the continent. Analyses of archaeological faunal assemblages are essential for elucidating diet breadth but cannot fully resolve it since the compositions of these assemblages may be biased by factors related to preservation, animal processing, and recovery (3). In contrast, stable isotope analysis of human remains provides a powerful tool for directly quantifying ancient diets and revealing the food sources actually consumed by individuals (4). The remains of two infants from the Upward Sun River (USR) site (Fig. 1), the earliest human remains in eastern Beringia, offer a rare opportunity to investigate the diet of ancient Native Americans using stable isotope analysis.
Two female infants were recovered in a single burial dating to ~11,500 calibrated years before present (cal yr. BP) at the USR site in interior Alaska (5). The older individual (Xachiteeaanenh Teede Gaay or USR1) was a newborn infant (~3 to 4 weeks), and the younger individual (Yekaanenh Teede Gaay or USR2) was a late-term prenate (5). Genomic analyses indicate that the two infants are the first known members of one of the two basal branches of Native Americans, termed Ancient Beringians (6). These infants belong to different maternal lineages (C1b and B2 haplogroups, respectively) (7) and provide two independent windows into the paleodiets of the mothers and, more broadly, of Ancient Beringians at the Pleistocene-Holocene transition. USR and the infants are assigned to the Denali Complex, a widespread cultural group present in eastern Beringia from 12,500 to 6000 cal yr. BP (5).
Here, we present multitissue stable isotope analyses of the USR infants to model the diets of their mothers based on a suite of comparative archaeological faunal data from the region, including large terrestrial herbivores (bison and wapiti), small game (hare, ground squirrel, and grouse/ptarmigan), salmon, and freshwater whitefish (tables S1 to S4 and fig. S1). Terminal Pleistocene human remains from North America are extremely rare, and to date, no isotope-based dietary analyses using regional archaeofaunal specimens have been conducted for this time period.
To gain a holistic understanding of the diet, we analyzed multiple tissues, as they reflect different dietary components: (i) carbon and nitrogen stable isotope values (13C and 15N values, respectively) of bulk bone collagen, (ii) 13C values of bone collagen single essential amino acids (EAAs) (compound specific isotope analysis), (iii) 13C values of bone bioapatite, and (iv) 13C values of tooth enamel. Dietary protein sources are reflected in (i) and (ii), and whole diet carbon sources (carbohydrates, lipids, and proteins) are reflected in (iii) and (iv) (4, 8, 9). Because USR2 died before birth, and USR1 likely died shortly after birth, the isotopic composition of their tissues will reflect maternal diets during pregnancy while the tissues were forming (10, 11), although we consider the possible effects of gestation and/or breastfeeding on isotope values.
Season of death for both infants is estimated as early August based on multiple proxies, including seasonally sensitive plant and animal taxa (berries, immature and mature ground squirrel, and salmon) (table S5). To determine the dietary window represented by the bone tissues of USR1 and USR2, we developed models of fetal bone collagen and mineral turnover rates.
Our models of fetal bone tissue turnover indicate rapid turnover rates for bone collagen and mineral during gestation (table S6 and fig. S2). For example, for an infant at age 40 gestational weeks, around 17% of existing bone collagen was formed in the previous week (Fig. 2). Applying the turnover models to the USR infants shows that the majority (~70%) of bone collagen was synthesized in the last 5 to 8 weeks of the infants lives (table S7). On the basis of the estimated time of death, the infants bone tissues would largely reflect maternal diets over the summer.
For a prenate at age 33 gestational weeks (GW), 64% of existing collagen was formed in the previous 4 weeks (30th to 33rd week), while for a newborn (40 GW), 55% of bone collagen was formed in the previous 4 weeks. For a neonate at 3 postnatal weeks (PW), 26% of bone collagen was formed in the previous 4 weeks, and 68% was formed in the previous 8 weeks. Estimates are based on a new model of fetal bone collagen turnover (tables S6 and S7); postnatal estimates are based on a previously published model (15).
We assume that bone collagen formed in utero reflects mothers diet since the fetus derives its nutrition from the maternal bloodstream via the placenta. However, we recognize that the question of whether mother and fetal bone collagen isotope values are identical is unresolved (12). We addressed this uncertainty by incorporating variance parameters for trophic discrimination factors (TDFs; the change in isotope values between food sources and consumer tissues) into our mixing models, as well as by examining the sensitivity of our mixing models to varied TDFs.
We analyzed the bulk bone collagen isotope data of the humans and ancient regional fauna both graphically (13) and using a Bayesian multisource mixing model (MixSIAR) (14) to estimate dietary protein sources for the mothers of USR1 and USR2 (Figs. 3 and 4 and tables S8 and S9). The USR infants have identical bone collagen 13C values (18.4), but USR1 has a slightly higher 15N value (9.1) compared to USR2 (8.7) (table S2), possibly reflecting the onset of nursing, which leads to 15N enrichment in tissues (11). Using the 15N value for USR2 as a prenursing baseline, we estimate that the slight bone collagen 15N enrichment of 0.4 would be achieved between 3 to 4 weeks of nursing, assuming normal bone growth and remodeling for USR1 and a maximum 15N enrichment of 3.0 through breastfeeding (11, 15).
Small game is an average of hare, ground squirrel, and grouse/ptarmigan. (A) Raw bone collagen 13C and 15N values. Salmon (P/H) includes specimens dating to the terminal Pleistocene/early Holocene; Salmon (LH) includes specimens dating to the Late Holocene. (B) Isospace plot showing mean faunal 13C and 15N values corrected for TDFs is as follows: 13CTDF-corrected = 13Ccollagen + 1.1 (0.2) and 15NTDF-corrected = 15Ncollagen + 3.8 (1.1) (66). Source error bars show 1 SD [combined source and TDF SD calculated following (71)]. Salmon includes P/H and LH specimens.
Estimated contributions (%) of food sources to maternal diets were determined using a five-source (bison, wapiti, small game, salmon, and whitefish), two-biotracer (13Ccollagen and 15Ncollagen) mixing model in MixSIAR. Terrestrial is an a posteriori aggregation of bison, wapiti, and small game. (A) Box plots showing median (center line), 50% credible interval (CI) (box edges), and 95% CI (error bars) for estimated contributions. (B) Posterior distributions (colored) superimposed on prior distributions (gray) of source contributions for USR1 (darker color) and USR2 (lighter color). Prior is the uninformative Dirichlet (equal weights for all five original sources).
Biplots of infant and faunal 15N and 13C values (Fig. 3, A and B) show that the infant isotope values fall within the dietary mixing space bounded by the faunal isotope values (after correcting for TDFs), demonstrating that their isotopic compositions can be explained by a mixture of the selected food sources in maternal diets (13). The infant isotope values are located close to those of the TDF-corrected terrestrial fauna (bison, wapiti, and small game), indicating that one or more of these resources contributed substantially to maternal diets. However, the infant 13C values are higher than those of the terrestrial sources (and the whitefish), indicating that a 13C-enriched source, salmon, must have contributed to the diets of both mothers. Furthermore, a plot that includes rarer secondary fauna shows that no other source exhibits the combination of 13C and 15N enrichment necessary to explain the infant isotopic signatures in the absence of salmon (fig. S3).
Bayesian mixing model results for both USR1 and USR2 indicate that although their mothers derived most of their dietary protein from terrestrial resources, salmon also contributed a substantial proportion, with negligible contributions from freshwater fish (Fig. 4A and table S9). Mean dietary contribution estimates for USR1 are 62 8% terrestrial (an a posteriori aggregation of bison, wapiti, and small game), 32 6% salmon, and 6 5% freshwater whitefish. Results are very similar for USR2, with mean estimates of 65 8% terrestrial, 30 7% salmon, and 5 4% freshwater whitefish. Bayesian 95% credible intervals (CIs) for each of the three sources indicate that there are no credible solutions composed of solely terrestrial sources (table S9). All solutions within the 95% CI also contain at least 16% salmon, although there are solutions in this interval that include no whitefish. Mixing model performance indicators are strong when considering the three major resource categories (terrestrial, salmon, and whitefish), with relatively constrained proportional contribution estimates and narrow, unimodal posterior distributions that diverge from the prior distributions, indicating that diet contribution estimates are robust and informed by the isotope data (Fig. 4, A and B) (14, 16). However, mixing model output cannot clearly resolve the relative importance of the discrete terrestrial resources, as there is a high degree of uncertainty for contribution estimates for bison, wapiti, and small game (table S9 and fig. S4).
Given the uncertainty in the estimate of the nitrogen stable isotope TDF (15N), we examined the sensitivity of mixing model contribution estimates to different magnitudes of 15N. We varied 15N in increments of 1 around our main model choice of 15N = 3.8, spanning 2.8 to 5.8. We found that the mean contribution estimates for terrestrial, salmon, and whitefish sources are fairly robust to changes in 15N (table S10). For example, for each 1 increase in 15N, the estimated mean salmon contribution decreases by only about five percentage points.
We measured the stable carbon isotope composition of several bone collagen EAAs (table S11). Because EAAs cannot be synthesized by animals, their carbon is routed from dietary protein with minimal modification and reflects the stable carbon isotopic composition of primary production sources at the base of the food web (17). In addition, individual amino acid 13C values can be normalized to control for baseline variation in isotope values, allowing comparisons of diets among human groups from different regions or time periods (18). Here, we use two normalized EAA dietary markers, 13CVal-Phe and 13CLys-Phe, calculated from published data, to discriminate between aquatic and terrestrial consumers (Fig. 5 and table S12) [modified from (18, 19)]. There is clear separation among consumers, with higher values of 13CVal-Phe and 13CLys-Phe for marine and freshwater consumers compared to terrestrial consumers, with no overlap in values. These differences parallel those found in algae and plants, which are major primary producers at the base of aquatic and terrestrial food webs, respectively. The USR infant values fall between those of the terrestrial and marine consumers, as expected if maternal diets were based on a combination of terrestrial mammals and salmon.
Comparative values were calculated from published data (table S12) (1719, 7274). 13CVal-Phe = 13Cvaline 13Cphenylalanine and 13CLys-Phe = 13Clysine 13Cphenylalanine. Values are also shown for primary producers. PP, primary producer; FWPC, freshwater protein consumer; MPC, marine protein consumer; TPC, terrestrial protein consumer.
We also measured the stable carbon isotope composition of USR1 bone bioapatite, which is 15.5. Bioapatite sample quality was evaluated using Fourier transform infrared spectroscopy (20, 21), and sample quality indictors including carbonate-to-phosphate ratio (0.28) and infrared splitting factor (2.6) are within the parameters of well-preserved archaeological samples (21). We used 13Cbioapatite and 13Ccollagen values in tandem to provide additional insights into maternal diet. Experimental evidence from animals fed controlled diets shows that 13Ccollagen and 13Cbioapatite distributions form two distinct regression lines depending on whether protein is derived from (i) C3 terrestrial sources or from (ii) marine or C4 terrestrial sources (22). Because C4 plants are extremely rare in the subarctic (23), we consider the second regression line as reflecting a marine diet. USR1 falls between the two protein lines but nearer to the C3 terrestrial protein line (fig. S5), in strong agreement with mixing model results based on bone collagen 13C and 15N values.
The spacing between 13Cbioapatite and 13Ccollagen values (13Cbioapatite-collagen) for USR1 is 2.9, which indicates that the 13C value of the whole diet is lower than that of dietary protein (9). For diets based on terrestrial animals, 13Cbioapatite-collagen is predicted to narrow as the 13C-depleted lipid fraction of the diet increases, with a predicted spacing of around 3 when lipid comprises 50% of macronutrients (24). Overall, the USR spacing suggests a high animal lipid content, which is consistent with diet inferences based on our other proxies, and further suggests substantial consumption of large mammals, which have a much higher percentage of whole body fat than small mammals (25).
Tooth enamel forms incrementally and is not remodeled, so isotopic analysis of serial samples from this tissue can detect changes in diet over the course of tooth formation (26). For USR1, we sampled enamel from the upper second deciduous incisor (di2), which begins forming around the 17th gestational week (27). Serial samples of enamel from di2 produced 13C values of 14.2 for the incisal section (formed earlier, ~February/March to May) and 13.4 from the cervical section (formed later, ~May to August). The increase in USR1 enamel 13C values over time of nearly 1 suggests the incorporation of 13C-enriched salmon in maternal diet during later development, consistent with the timing of modern salmon runs in the Tanana basin (28).
Zooarchaeological analyses of contemporaneous sites in the Tanana basin (29, 30) and USR-specific hearth sediment isotopic analyses (31) provide independent proxies for Ancient Beringian paleodiets. Very large mammals (primarily bison and wapiti) are found at all Denali Complex sites with fauna (n = 16; 100% ubiquity), while small mammals (e.g., ground squirrel and hare), birds (terrestrial and waterfowl), and fish are found at fewer sites (29, 44, and 25% ubiquity, respectively) (tables S13 and S14) (29, 30). Among ungulates, bison (56% occurrence) and wapiti (31%) are most common, followed by caribou and sheep (19%) and moose (6%). Some short-term Denali hunting camps, such as Gerstle River (32), are dominated by bison and wapiti and a narrow range of weapon technology, while Denali residential base camps, such as USR and Broken Mammoth, have a much wider range of faunal taxa (table S14) (5, 33).
Paleoindian use of salmon has only recently been confirmed in a Denali site, at USR component 3 (34). We report here the identification of chum salmon (Oncorhynchus keta) remains from a second site (XBD-318) in the Tanana basin. The salmon specimen was recovered from an archaeological component buried in loess (35) and directly dated to 12,680 to 12,770 cal yr. BP (UGAMS-26403; 10,830 40 BP). The specimen has been confirmed as an anadromous chum salmon through DNA and isotopic analyses (tables S1 to S4).
There is also strong agreement between our estimated terrestrial and salmon contributions to diet based on bone collagen 13C and 15N values and those based on carbon stable isotope analyses of fatty acids in sediments from multiple hearths at USR component 3 (table S15 and fig. S6) (31). Collectively, the regional zooarchaeological record and hearth chemical profiling from the burial component support terrestrial subsistence dominated by bison and wapiti, with some use of salmon, freshwater resources, and small game.
These stable isotope-based paleodiet models provide unique windows into the diets of two Ancient Beringian women over a single spring and summer period 11,500 years ago, providing a link between the broad-scale subsistence patterns observed in the archaeological record over millennia and the short-term foraging decisions made by individuals. Our results indicate that over a summer season, the mothers of USR1 and USR2 obtained a majority of dietary protein from terrestrial food resources but salmon also contributed substantially, while freshwater fish were of minor importance. Salmon were probably only available in the last 4 to 6 weeks of the infants lives, corresponding to the time period during which the bulk of bone collagen was forming, and the tooth enamel data from USR1 indicate that there was increased consumption of a 13C-enriched source, likely salmon, between the spring and summer seasons.
Current models for terminal Pleistocene subsistence and mobility in eastern Beringia indicate that the availability of terrestrial resources, and particularly the availability of megafauna, was the principal conditioning factor for making economic and land use decisions (30). Our paleodiet models do not indicate either strictly megafaunal specialists or generalized broad-spectrum foragers. Collectively, these diet models combined with archaeological data from Denali Complex large mammal hunting stations (32) and residential base camps such as USR (5) provide a more holistic perspective on Ancient Beringian diets. We have shown that several independent proxies of diet, including isotopic analyses of multiple human tissues and hearth sediment fatty acids (31), along with regional zooarchaeological evidence (30), support the conclusion that Ancient Beringian diets were complex and seasonally structured. While the diet models produced here support the primacy of terrestrial resources to Ancient Beringian diets, the isotopic evidence for substantial inputs from salmon suggests that this resource too was important in shaping mobility and settlement systems.
Salmon were historically a critical resource for Athabascan populations in interior Alaska (36), and they continue to be important today. An extensive diet survey of modern indigenous Alaskan populations (37) found that Athabascan groups in the Tanana region derive 24% of their dietary protein from salmon, a figure very similar to our estimate for the salmon contribution to Ancient Beringians living in the area 11,500 years ago.
All human and faunal bone samples analyzed in this study are presented and described in table S1. All specimens came from existing collections permanently curated at the University of Alaska Museum of the North or under active investigation at research laboratories.
USR infants. Excavation and analyses of the USR infant remains were conducted under a Memorandum of Agreement (MOA) signed by the State of Alaska (the land owner), the National Science Foundation (the lead federal agency), the Healy Lake Tribal Council (the federally recognized tribal authority), and the Tanana Chiefs Conference (the regional nonprofit tribal consortium). An amendment to the MOA was signed by all parties to allow for destructive analysis of skeletal remains for the purposes of diet reconstruction through stable isotope analyses and assessment of genetic relationships through DNA analyses. We are grateful for the support and cooperation of all parties.
Osteological and genetic studies of the USR infants have been published previously (57). Mitochondrial DNA sequences show that the infants belong to two separate mitochondrial lineages (B2 and C1b) and, thus, are not maternally related (7). Whole-genome sequence data indicate that relatedness between the USR infants is within the range of half siblings or first cousins, and they confirm that the two infants are female (6).
Osteometric estimates of age at death are around birth to 6 weeks for USR1 and around 33 weeks in utero for USR2 (5). The age estimate based on tooth development is slightly higher for USR1 (~12 postnatal weeks). The discrepancy between the osteometric and dental ages may have resulted from differences between Ancient Beringians and the modern European-derived individuals used to the develop the age schedules, or bone growth could have lagged behind chronological age for USR1, although no skeletal anomalies or pathologies are apparent (5). The uncertainty ranges surrounding the point age estimates are larger for the dental methods (~4 to 13 weeks) than for the osteometric methods (~2 weeks) (5, 38). Given that the one-sigma uncertainty ranges for the two methods overlap at around age 3 to 4 weeks, we suggest that this is a reasonable estimate of the age at death for USR1.
Single middle ribs from USR1 and USR2 were selected for bone collagen extraction. The rib from USR1 produced sufficient material to also allow bioapatite extraction. In addition, enamel from a single tooth (deciduous right maxillary lateral incisor) from USR1 was serially sampled for 13C analysis. Serial enamel sampling was attempted for USR2, but the samples were too small for analysis.
Faunal remains. We selected faunal taxa based on their presence in the USR component 3 faunal assemblage (directly associated with the infants) and/or their ubiquity in regional terminal Pleistocene/early Holocene faunal assemblages. Faunal remains directly associated with the USR infants are generally too burned to be good candidates for collagen extraction and stable isotope analysis. Therefore, we selected faunal remains from other archaeological sites in the Tanana basin of central Alaska to encompass the suite of potential faunal resources used by USR inhabitants and to capture food source isotopic variation (table S1). We used molecular methods (including DNA and/or protein fingerprinting) to confirm or refine the taxonomic identifications of all salmon and nearly all ungulates (tables S1, S3, and S4 and data file S1). To control for interlaboratory and interstudy variation in measurements of 13Ccollagen and 15Ncollagen (39), all faunal specimens were analyzed specifically for this study using the same collagen extraction method and, except for a single specimen, using the same stable isotope laboratory (table S2).
We considered two sets of faunal food sources: (i) primary fauna, which includes only those fauna confirmed in USR component 3 (directly associated with the infants) and/or fauna that are ubiquitous in terminal Pleistocene/early Holocene faunal assemblages in the Tanana basin (bison, wapiti, small game, salmon, and whitefish); and (ii) secondary fauna, which includes taxa that are not confirmed in USR Component 3 but that occasionally occur in the faunal record for this time period (caribou, sheep, and waterfowl) (tables S13 and S14) (5, 30). Additional details on faunal sample composition are found in the Supplementary Materials.
DNA extraction. All prepolymerase chain reaction (PCR) activities were conducted in the ancient DNA facility at the Laboratories of Molecular Anthropology and Microbiome Research at the University of Oklahoma. This facility is a dedicated workspace for processing aged, degraded, and/or low copy number DNA samples. Precautions aimed to minimize and monitor the introduction of contamination are practiced in the laboratory.
Approximately 50 mg or less of bone material was subsampled from each specimen. The subsamples were submerged in 6% (w/v) sodium hypochlorite for 4 min (40). The sodium hypochlorite was poured off, and the samples were quickly submerged in DNA-free water twice.
The bone samples were transferred to 1.5-ml tubes, to which aliquots of 500 l of 0.5 M EDTA were added, and the tubes gently rocked at room temperature for >48 hours. An extraction negative control, to which no bone material was added, accompanied each batch of extractions, typically in a ratio of 1:7 with the samples.
DNA was extracted following the method described by Kemp et al. (41). Ninety microliters of proteinase K (Bio Basic catalog no. 32181) at a concentration of 1 mg/30 l (or >20 U/30 l) was added to each sample, and the tubes were incubated at 64 to 65C for 3 hours. Following proteinase K digestion, the tubes were centrifuged at 15,000 rpm for 1 min to pellet any undigested bone, dirt, and/or sludge. All centrifugation steps in this study were conducted with an Eppendorf centrifuge 5424. The liquid was carefully moved to a new 1.5-ml tube, to which 750 l of 2.5% resin (i.e., 2.5% celite in 6 M guanidine HCl) and 250 l of 6 M guanidine HCl were added. The tubes were vortexed multiple times over approximately a 2-min period.
Promega Wizard minicolumns were attached to 3 ml of Luer-Lok syringe barrels (minus the plunger) and placed on a vacuum manifold. Three milliliters of DNA-free water was first pulled across the columns with the intent to wash away potential contaminating DNA. The DNA/resin mixture was subsequently pulled across the columns. The silica pellet on the minicolumn was then rinsed by pulling 3 ml of 80% isopropanol across the columns.
The minicolumns were then placed in new 1.5-ml tubes and centrifuged at 10,000 rpm for 2 min to remove excess isopropanol. The minicolumns were transferred to new 1.5-ml tubes. Fifty microliters of DNA-free water heated to 64 to 65C was added to the minicolumns and left for 3 min before centrifugation of the tubes for 30 s at 10,000 rpm. This step was repeated, amounting to 100 l of extracted DNA. Ten microliters of the full concentration eluates and extraction negative controls were diluted 1:10 with water and used as described below [under standard PCR, rescue PCR, and PCR buffer enhancer P (PEC-P)].
Inhibition test and repeat silica extraction. The remaining volumes of the full concentration DNA eluates were tested for the presence of PCR inhibitors following the rationale of Kemp et al. (41) using a turkey collective as the ancient DNA positive control (see their figure 1). DNA recovered from seven or more archaeological turkey (Meleagris gallopavo) bones (42) was pooled together to make the turkey collective. The choice to pool these individual extractions was made with the intention to reduce variation between turkey DNA eluates in both endogenous mitochondrial DNA copy number and possible inhibitors coextracted with the turkey DNA. Before they are used in experiments, each turkey collective was demonstrated to PCR amplify consistently (in six or more amplifications), hence serving as a positive control.
Fifteen microliters of PCRs were conducted to amplify a 186base pair portion of turkey displacement loop using the primers T15709F and T15894R described by Kemp et al. (42). The components of these PCRs were as follows: 1 Omni Klentaq Reaction Buffer (including a final concentration of 3.5 mM MgCl2), 0.32 mM deoxynucleotide triphosphates (dNTPs), 0.24 M of each primer, 0.3 U of Omni Klentaq LA polymerase, and 1.5 l of turkey collective DNA. These reactions were spiked with 1.5 l of potentially inhibited, full concentration DNA eluates recovered from the samples under investigation here. The extraction negative controls were also tested for inhibitors in this manner. These PCRs were run in parallel with reactions that contained only turkey collective DNA (i.e., were not spiked). These reactions served as positive controls and allowed us to preclude PCR failure from contributing to our results. PCR negatives also accompanied each round of amplification, allowing us to monitor for possible contamination. Additional positive controls of modern turkey DNA were added in the post-PCR laboratory only before initiating amplification, serving as another check for possible PCR failure. Following denaturing at 94C for 3 min, 60 cycles of PCR was conducted at 94C for 15 s, 60C for 15 s, and 68C (note that this is the optimal extension temperature for Omni Klentaq LA polymerase) for 15 s. Last, a 3-min extension period at 68C was conducted before bringing the reactions to 10C.
If the turkey collective failed to amplify when spiked with any given ancient DNA eluate, then we considered the eluate to be inhibited. In the case that spiking the ancient DNA permitted amplification of the turkey collective DNA, we consider that DNA eluate to be inhibitor free.
Full concentration eluates deemed to be inhibited using the test outlined above were subjected to repeat silica extraction (41). To the remaining volume of the eluate, 750 l of 2.5% resin and 250 l of 6 M guanidine HCl were added. The samples were vortexed numerous times over a 2-min period. The extraction then followed procedures described above under DNA extraction, except that the volume used to elute the DNA from column matched the volume being repeat silica extracted. For example, if the starting volume was 87 l, then 43.5 l of DNA-free water heated to 65C was added to the minicolumns and left for 3 min before centrifugation. This step was repeated twice for a total volume of 87 l.
These repeat silica eluates were tested again for inhibition, as described above. Those still deemed to be inhibited were once again repeat silica extracted and tested again for inhibition. This was carried out until all full concentration eluates were deemed to be uninhibited.
Standard PCR, rescue PCR, and PEC-P. All of the full concentration, uninhibited eluates and the original 1:10 dilutions of the full concentration eluates were subject to three forms of PCR. First, standard PCRs contained 1 Omni Klentaq Reaction Buffer, 0.32 mM dNTPs, 0.24 M of each primer, 0.3 U of Omni Klentaq LA polymerase, and 1.5 l of template DNA. Second, rescue PCR at a 25% increase was carried out, as described by Johnson and Kemp (43). Rescue PCRs contained 1.25 Omni Klentaq Reaction Buffer (including a final concentration of 4.375 mM MgCl2), 0.4 mM dNTPs, 0.3 M of each primer, 0.375 U of Omni Klentaq LA polymerase, and 1.5 l of template DNA. Last, we used PEC-P. The composition of this enhancer cocktail is proprietary, and no safety data sheet is made available at the DNA Polymerase Technology website (www.klentaq.com/). However, Palmer et al. (44) used PEC-P to increase their success in species identification of archaeological remains of smelt and other small fishes. These PCR reactions contained 1 Omni Klentaq Reaction Buffer (including a final concentration of 3.5 mM MgCl2), 0.32 mM dNTPs, 0.24 M of each primer, 0.3 U of Omni Klentaq LA polymerase, 20% (v/v) PEC-P, and 1.5 l of template DNA. All three forms of PCR were conducted as follows: (i) 94C for 3 min; (ii) 60 cycles of PCR at 94C for 15 s, the annealing temperature for 15 s (table S3), and 68C for 15 s; and (iii) a 3-min extension period at 68C before bringing the reactions to 10C.
Primers. Primers are listed in table S3. For salmonid species identification, we used previously described primers by Jordan et al. (45). Note that Jordan et al. (45) originally described their reverse primer in the wrong orientation. The corrected primers are OST12S-forward (5-GCTTAAAACCCAAAGGACTTG-3) and OST12S-reverse (5-CTACACCTCGACCTGACGTT-3). Other primers were designed in this study to differentiate moose (Alces alces), bison (Bison priscus), elk (Cervus canadensis), Dall sheep (Ovis dalli), and caribou (Rangifer tarandus).
Sequencing results. Sequencing results are shown in table S4, and species identification is noted in table S1. Samples 16.179, 16.180, 16.181, 16.182, 16.184, and 17.276 were identified as bison (B. priscus) with repeatable results. DNA from sample 16.185, identified as bison based on morphology and as Bison/Bos based on ZooMS results, failed to amplify despite repeated attempts.
Sample 17.284 was identified as a caribou (R. tarandus) from the amplicon produced with primers Art3F/Art3R. Despite repeated PCRs, this observation was not repeatable. However, note that this sample was believed to be a caribou based on morphology.
Samples 15.301, 16.109, and 17.106 were identified as chum salmon (O. keta), as described by Jordan et al. (45), exhibiting the following mutations relative to the rainbow trout (Oncorhynchus mykiss), reference sequence (DQ288271.1): 660T and 713T. Samples 17.109, 17.110, and 17.112 were also identified as chum salmon with an additional derived mutation of a thymine (T) deletion at nucleotide position 668. Replication was possible for these six salmonid specimens. A single amplicon from sample 17.111 revealed this specimen to be a chum salmon with a T deletion at nucleotide position 668. Sequences from two additional amplicons from this sample showed additional but different mutations, likely attributable to postmortem damage.
Samples 16.171, 16.175, and 16.176 were identified as elk (C. canadensis) with primers ElkCOIF/ElkCOIR, matching the reference sequence (JF443209.1). Only a single amplicon was produced for sample 16.171, despite repeated attempts at amplification. Three additional amplicons produced from sample 16.175 differed from the reference sequence by different mutations, likely as a product of postmortem damage. A second amplicon sequenced from sample 16.176 also showed signs of damage with mutations at nucleotide positions 230T and 234T. Samples 16.171, 16.175, and 16.176 were identified as elk by morphological assessment and as Cervidae by ZooMS analysis. DNA from samples 16.168, 16.169, 16.170, 16.172, 16.173, 16.174, and 16.177 failed to amplify.
A subsample of approximately 1 to 2 mg of bone collagen (below) was resuspended with 50 mM ammonium bicarbonate and digested with 0.4 g of sequencing grade trypsin (Promega, UK) overnight at 37C. Digests were then acidified to 0.1% trifluoroacetic acid (TFA) and fractionated into 10 and 50% acetonitrile (in 0.1% TFA) before being dried down to completion by centrifugal evaporation and resuspension in 10 l of 0.1% TFA. One microliter of resuspended peptide mixture was then spotted onto a stainless steel matrix-assisted laser desorption/ionizationtime-of-flight (MALDI-TOF) plate along with an equal volume of -cyano hydroxycinnamic acid matrix (10 mg/ml) in 50% ACN (acetonitrile)/0.1% TFA and allowed to air dry. MALDI-TOF mass spectrometry was carried out using a Bruker Ultraflex II with 2000 laser acquisitions over the range mass/charge ratio of 700 to 3700, and the resultant spectra were compared with reference spectra published previously (46). Results are shown in table S1, fig. S1, and data file S1.
All carbon and nitrogen stable isotope measurements are expressed in delta notation (as 13C and 15N, respectively) relative to internationally accepted standards as follows: = (Rsample Rstandard)/Rstandard, where R is the ratio of the heavy to light isotope (e.g., 13C/12C) (47); by convention, this quantity is multiplied by 1000 to report the result in parts per thousand (). The international standard for carbon is Vienna Pee Dee belemnite (VPDB) and that for nitrogen is atmospheric N2 (AIR). The standard reference materials used to calibrate raw isotopic measurements to the internationally accepted scales are described for each laboratory below.
Bone collagen (13C and 15N values). All human and faunal bone collagen extractions were conducted in the same laboratory (Laboratory of Environmental Archaeology, University of Alaska Fairbanks) using the modified Longin (48) protocol described previously (34). Briefly, powdered bone was demineralized in HCl (0.5 M), followed by treatment in NaOH (0.1 M), and gelatinization at 70C in dilute HCl (0.001 M, pH 3). Bone collagen samples were submitted to the Washington State University Stable Isotope Core Laboratory for carbon and nitrogen stable isotope measurement on a Thermo Finnigan Delta Plus XP continuous flow isotope ratio mass spectrometer, coupled to a Costech elemental analyzer (ECS 4010). The carbon and nitrogen stable isotope compositions were calibrated relative to VPDB and AIR, respectively, using at least two internal standards, which had been previously calibrated against internationally certified standards (table S16). In addition, all sample sequences included the same quality control check standard, casein (B2155 Elemental Microanalysis), as a check of the normalization. Precision was calculated as the pooled SD of all repeated measures of calibration and check standards over the relevant analytical runs following Szpak et al. (49) and was 0.14 for both 13C and 15N. One extracted bone collagen sample (#17.260) was analyzed by the Center for Applied Isotope Studies at the University of Georgia. This laboratory calibrated relative to VPDB and AIR using two in-house standards, spinach (13C = 27.22 and 15N = 0.19) and bovine (13C = 17.53 and 15N = 8.14), which had been previously calibrated to National Institute of Standards and Technology standards.
All bone collagen samples used in this study met well-accepted quality standards: %N > 5%, %C > 13%, an atomic C/N ratio of 2.9 to 3.6, and an collagen yield of >1% (table S2) (5053). Further, our 13C and 15N values for ungulates are generally comparable to those previously reported for archaeological specimens in the region and elsewhere in Alaska, although there is variation among studies (5457). We further note that the stable carbon isotope composition of our salmon specimens, as in other archaeological and modern Pacific salmon samples, is relatively depleted in 13C compared to many marine fishes, likely due to the offshore feeding location of many salmon species (5862).
Bone collagen EAAs (13C values). We measured amino acid 13C values using gas chromatographycombustionstable isotope ratio mass spectrometry (GC-C-IRMS) in the Alaska Stable Isotope Facility (ASIF) at the niversity of Alaska Fairbanks. To prepare samples for analysis, we hydrolyzed dried collagen (~1 mg) using 1 ml of HCl (6 M) at 110C for 20 hours and dried them under N2. We derivatized the amino acids to N-acetyl methyl esters (NACMEs) for analysis by GC-C-IRMS (63). First, we methylated the amino acids with acidified methanol, prepared by the addition of acetyl chloride to methanol (1:6, v/v) in an ice bath. The methylation was completed during 75C incubation for 1 hour. Samples were then dried down under N2 and acetylated with the addition of acetic anhydride, triethylamine, and acetone (1:2:5, v/v/v) and an incubation at 60C for 10 min. The NACMEs (i.e., the derivatized amino acids) were dried down and purified with a phosphate buffer wash [1 M potassium phosphate and 1 M sodium phosphate (pH 7)], extracted with chloroform, and, again, dried down under N2. Last, the NACMEs were dissolved in ethyl acetate. A mix of amino acid standards with known 13C values was prepared alongside samples, and an internal standard (norleucine) was added to standards and samples. Amino acid NACMEs were injected onto a VF-35ms column (Agilent) in a TRACE 1310 GC interfaced with an Isolink II (Thermo Scientific), combusted into CO2 gas, and the carbon isotope ratios of individual NACME peaks were measured on a Delta V Plus IRMS (Thermo Scientific). The 13C calculation was based on NACME peak integration, which was performed by the program Isodat (version 3.0, Thermo Scientific). Peak integration was visually assessed for correct identification, width and background assignment, and adequate baseline separation between peaks. We measured the 13C in six EAAs: isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), threonine (Thr), and valine (Val). The 13C values of individual amino acids are measured relative to the 13C values of standard amino acids; these are internal (in-house ASIF) standards, and their 13C, before derivatization, are given in table S17. Internal (rather than international) individual amino acids are used to account for the potential influence of slightly different fractionations that can occur between batches of sample derivatizations and to allow for the correction of carbon added during the derivatization process (17, 63). Analytical precision was <0.3 for each of the amino acids in both the standards (<0.2) and samples (with the exception of phenylalanine that was <0.8 in samples and <0.2 in standards).
Bone bioapatite (13C values). The USR1 bone sample yielded sufficient bulk bone powder to allow both collagen and bioapatite extraction, but the USR2 bone sample was exhausted after collagen extraction. Bone bioapatite for USR1 was extracted following Garvie-Lok et al. (20) with minor modifications. Briefly, organics were removed from cleaned, powdered bone by soaking in 2% NaOCl for 48 hours (with a refresh at 24 hours), followed by rinsing and removal of contaminating carbonate by treatment in 0.1 M acetic acid (unbuffered) for 24 hours, and followed by a final rinsing and freeze drying for 48 hours. Bioapatite samples were submitted to the University of California Santa Cruz Stable Isotope Laboratory for carbon stable isotope measurement by acid digestion using a Thermo Scientific Kiel IV carbonate device interfaced to a Thermo Scientific MAT 253 isotope ratio mass spectrometer. Carbon stable isotope composition was calibrated relative to VPDB using NBS-18 and the internal standard CM12 (Carrara Marble; 13C = 2.05). The internal standard was previously calibrated against NBS-19 and NBS-18. Precision for 13C over the analytical run was calculated as defined above and was 0.11.
Tooth enamel bioapatite (13C values). Enamel from a single tooth (deciduous right maxillary lateral incisor) from USR1 was serially sampled for 13C analysis. Serial enamel sampling was attempted for USR2, but yields were too low for analysis. Because enamel forms incrementally and is not remodeled once formed, serial samples can be used to detect changes in diet over the course of tooth formation. Tooth enamel formation for the maxillary di2 begins at approximately 163 days before birth (~17 gestational weeks) (27). Assuming a time of death in early August, if USR1 died at birth (40 gestational weeks), then her di2 tooth enamel likely began forming in early March, whereas if USR1 lived for several weeks after birth, then the start of enamel formation would be pushed back accordingly (e.g., ~mid-February if USR1 lived for 3 weeks, ~mid-January if USR lived for 6 weeks, etc.). Enamel formation begins at the cusp of the developing tooth and proceeds toward the tooth cervix. The tooth was serially sampled in two locations, one (cuspal) reflecting earlier development (i.e., ~spring) and one (cervical) reflecting later development (i.e., ~summer).
To prepare enamel bioapatite, the tooth was mechanically cleaned to remove surface contaminants and then ultrasonically cleaned in deionized double-distilled water (DDH2O) for 30 min. Approximately 2.0 mg of cleaned enamel powder was drilled from the tooth. The samples were first treated in microcentrifuge tubes with 3% H2O2 for 15 min and then were rinsed three times with DDH2O water before being treated with 0.1 M CH3COOH for 15 min. Samples were rinsed three times with DDH2O water and then dried overnight in an oven at 60C. 13C values were measured by continuous-flow IRMS at IsoForensics in Salt Lake City, Utah using a GasBench (Thermo Scientific) interfaced to an isotope ratio mass spectrometer (Thermo Finnigan MAT 253). The carbon stable isotope composition was calibrated relative to VPDB using NBS-19 and LSVEC. Precision for 13C over the analytical run was calculated as described above and was 0.10.
To estimate proportional contributions of food sources to the diets of the USR infants mothers, we used MixSIAR (14), a Bayesian stable isotope mixing model framework available as an open-source package in R (64).
Food source selection and aggregation. For diet estimation using stable isotope mixing models, the number of diet sources should be kept as low as possible without excluding important sources (14, 16, 65). Bayesian mixing models assume that all sources included in the model contributed to diet, so it is important to include only sources that were actually consumed (65). For the MixSIAR analyses, we used the five sources (bison, salmon, small game, wapiti, and whitefish) described above as primary fauna.
Trophic discrimination factors. We used the following collagensource-to-collagenconsumer TDFs (with associated SDs) following Bocherens et al. (66): 13Cconsumer collagensource collagen = 1.1 0.2 and 15Nconsumer collagensource collagen = 3.8 1.1. We further assumed that fetal and neonate bone collagen isotope values reflect those of the mothers diet (after adjusting for the standard TDFs) but with caveats. The assumption is supported by limited isotopic analyses of accessible proteinaceous hard tissues (fingernails) in living mother/infant pairs, which show that newborn and mother fingernail 15N and 13C values are similar (11, 67). A study of hair from newborn-mother pairs likewise found a negligible difference (+0.4) for 13C values but a +0.9 enrichment for 15N (68). Studies comparing archaeological adult and infant bone collagen isotope values are mixed for 15N values, with a meta-analysis by Reynard and Tuross (12) showing neonate bone collagen 15N values both above and below the adult female mean.
Addressing uncertainty in the 15N TDF (15N). To address uncertainty in 15N between consumer and source collagen, as well as between mothers diet and fetal/neonate bone collagen, (i) we include a variance parameter in our 15N our isotope mixing models, and (ii) we explore the sensitivity of model mean proportional contribution estimates to various magnitudes of 15N. We set 15N variance at 1.2, the squared SD of 1.1 found by Bocherens et al. (66) in their meta-analysis of prey-to-predator, collagen-to-collagen offsets in 15N values. We varied 15N in increments of 1 around our main model choice of 3.8, from 2.8 to 5.8, to approximate the range of reported nitrogen isotope TDFs for humans and other mammals (69, 70).
We used the Bayesian multisource mixing model MixSIAR (14) to estimate dietary protein sources for the mothers of USR1 and USR2. MixSIAR models were run separately for USR1 and USR2. Table S8 provides the consumer and food source isotope values, TDFs, and associated uncertainties included in the models. Markov chain Monte Carlo parameters were as follows: (i) normal run: chain length = 100,000, burn-in = 50,000, thinning = 50, and chains = 3; (ii) alpha.prior: Dirichlet prior (default, 1; uninformative); and (iii) error structure: residual error: FALSE (not included) and process error: TRUE (included) (note that this is the only error structure appropriate for fitting a single consumer) (14).
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Ancient Beringian paleodiets revealed through multiproxy stable isotope analyses - Science Advances
5 rice-based protein-rich salads for quick weight loss – Times of India
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This salad is made with multiple protein-rich ingredients such as tofu, sweet potato, leafy greens, green peas and peanut butter. It is as flavourful as it is healthy! Drizzle 1 tsp oil and salt to taste over 1 cup cubed sweet potatoes and bake for 30 minutes in the oven at 220 degrees Celsius. In a mixing bowl, add 2 cups chopped firm tofu and marinate in 2 tbsp soy sauce, 1 tsp garlic powder and tsp red chilli powder. Fry the tofu in 1 tsp oil for 5 minutes over medium heat in a pan until the colour changes. Then add boiled peas, cup cooked brown rice, 2 handfuls leafy greens of your choice, 2 finely sliced radishes (optional), fried tofu and baked sweet potatoes to a bowl. Make a dressing with 3 tbsp unsweetened peanut butter, 1 tbsp soy sauce, tsp maple syrup or honey, tsp apple cider vinegar or lemon juice, tsp garlic powder and salt & pepper to taste. Stir everything well and serve immediately.
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5 rice-based protein-rich salads for quick weight loss - Times of India