Aboriginal mitogenomes reveal 50,000 years of regionalism in Australia.

Aboriginal Australians represent one of the longest continuous cultural complexes known. Archaeological evidence indicates that Australia and New Guinea were initially settled approximately 50 thousand years ago (ka); however, little is known about the processes underlying the enormous linguistic and phenotypic diversity within Australia. Here we report 111 mitochondrial genomes (mitogenomes) from historical Aboriginal Australian hair samples, whose origins enable us to reconstruct Australian phylogeographic history before European settlement. Marked geographic patterns and deep splits across the major mitochondrial haplogroups imply that the settlement of Australia comprised a single, rapid migration along the east and west coasts that reached southern Australia by 49-45 ka. After continent-wide colonization, strong regional patterns developed and these have survived despite substantial climatic and cultural change during the late Pleistocene and Holocene epochs. Remarkably, we find evidence for the continuous presence of populations in discrete geographic areas dating back to around 50 ka, in agreement with the notable Aboriginal Australian cultural attachment to their country.

Impaired postprandial skeletal muscle vascular responses to a mixed meal challenge in normoglycaemic people with a parent with type 2 diabetes.

AIMS/HYPOTHESIS: Microvascular blood flow (MBF) increases in skeletal muscle postprandially to aid in glucose delivery and uptake in muscle. This vascular action is impaired in individuals who are obese or have type 2 diabetes. Whether MBF is impaired in normoglycaemic people at risk of type 2 diabetes is unknown. We aimed to determine whether apparently healthy people at risk of type 2 diabetes display impaired skeletal muscle microvascular responses to a mixed-nutrient meal. METHODS: In this cross-sectional study, participants with no family history of type 2 diabetes (FH-) for two generations (n = 18), participants with a positive family history of type 2 diabetes (FH+; i.e. a parent with type 2 diabetes; n = 16) and those with type 2 diabetes (n = 12) underwent a mixed meal challenge (MMC). Metabolic responses (blood glucose, plasma insulin and indirect calorimetry) were measured before and during the MMC. Skeletal muscle large artery haemodynamics (2D and Doppler ultrasound, and Mobil-O-graph) and microvascular responses (contrast-enhanced ultrasound) were measured at baseline and 1 h post MMC. RESULTS: Despite normal blood glucose concentrations, FH+ individuals displayed impaired metabolic flexibility (reduced ability to switch from fat to carbohydrate oxidation vs FH-; p < 0.05) during the MMC. The MMC increased forearm muscle microvascular blood volume in both the FH- (1.3-fold, p < 0.01) and FH+ (1.3-fold, p < 0.05) groups but not in participants with type 2 diabetes. However, the MMC increased MBF (1.9-fold, p < 0.01), brachial artery diameter (1.1-fold, p < 0.01) and brachial artery blood flow (1.7-fold, p < 0.001) and reduced vascular resistance (0.7-fold, p < 0.001) only in FH- participants, with these changes being absent in FH+ and type 2 diabetes. Participants with type 2 diabetes displayed significantly higher vascular stiffness (p < 0.001) compared with those in the FH- and FH+ groups; however, vascular stiffness did not change during the MMC in any participant group. CONCLUSIONS/INTERPRETATION: Normoglycaemic FH+ participants display impaired postprandial skeletal muscle macro- and microvascular responses, suggesting that poor vascular responses to a meal may contribute to their increased risk of type 2 diabetes. We conclude that vascular insulin resistance may be an early precursor to type 2 diabetes in humans, which can be revealed using an MMC.

Impaired postprandial adipose tissue microvascular blood flow responses to a mixed-nutrient meal in first-degree relatives of adults with type 2 diabetes.

Adipose tissue microvascular blood flow (MBF) is stimulated postprandially to augment delivery of nutrients and hormones to adipocytes. Adipose tissue MBF is impaired in type 2 diabetes (T2D). Whether healthy individuals at-risk of T2D show similar impairments is unknown. We aimed to determine whether adipose tissue MBF is impaired in apparently healthy individuals with a family history of T2D. Overnight-fasted individuals with no family history of T2D for two generations (FH-, n = 13), with at least one parent with T2D (FH+, n = 14) and clinically diagnosed T2D (n = 11) underwent a mixed meal challenge (MMC). Metabolic responses [blood glucose, plasma insulin, plasma nonesterified fatty acids (NEFAs), and fat oxidation] were measured before and during the MMC. MBF in truncal subcutaneous adipose tissue was assessed by contrast ultrasound while fasting and 60 min post-MMC. FH+ had normal blood glucoses, increased adiposity, and impaired post-MMC adipose tissue MBF (Δ0.70 ± 0.22 vs. 2.45 ± 0.60 acoustic intensity/s, P = 0.007) and post-MMC adipose tissue insulin resistance (Adipo-IR index; Δ45.5 ± 13.9 vs. 7.8 ± 5.1 mmol/L × pmol/L, P = 0.007) compared with FH-. FH+ and T2D had an impaired ability to suppress fat oxidation post-MMC. Fat oxidation incremental area under the curve (iAUC) (35-55 min post-MMC, iAUC) was higher in FH+ and T2D than in FH- (P = 0.005 and 0.009, respectively). Postprandial MBF was negatively associated with postprandial fat oxidation iAUC (P = 0.01). We conclude that apparently healthy FH+ individuals display blunted postprandial adipose tissue MBF that occurs in parallel with adipose tissue insulin resistance and impaired suppression of fat oxidation, which may help explain their heightened risk for developing T2D.NEW & NOTEWORTHY Adipose tissue blood flow plays a key role in postprandial nutrient storage. People at-risk of type 2 diabetes have impaired postmeal adipose tissue blood flow. Impaired adipose tissue blood flow is associated with altered fat oxidation. Risk of type 2 diabetes may be elevated by poor adipose tissue blood flow.

Femoral Heads from Total Hip Arthroplasty as a Source of Adult Hematopoietic Cells.

Normal human bone marrow cells are critical for studies of hematopoiesis and as controls to assess toxicity. As cells from commercial vendors are expensive, many laboratories resort to cancer-free bone marrow specimens obtained during staging or to umbilical cord blood cells, which may be abnormal or reflect a much younger age group compared to the disease samples under study. We piloted the use of femoral heads as an alternative and inexpensive source of normal bone marrow. Femoral heads were obtained from 21 successive patients undergoing elective hip arthroplasty. Mononuclear cells (MNCs) were purified with Ficoll, and CD3+, CD14+, and CD34+ cells were purified with antibody-coated microbeads. The median yield of MNCs was 8.95 × 107 (range, 1.62 × 105-2.52 × 108), and the median yield of CD34+ cells was 1.40 × 106 (range, 3.60 × 105-9.90 × 106). Results of downstream applications including qRT-PCR, colony-forming assays, and ex vivo proliferation analysis were of high quality and comparable to those obtained with standard bone marrow aspirates. We conclude that femoral heads currently discarded as medical waste are a cost-efficient source of bone marrow cells for research use.

Postprandial microvascular blood flow in skeletal muscle: Similarities and disparities to the hyperinsulinaemic-euglycaemic clamp.

Skeletal muscle contributes to ~40% of total body mass and has numerous important mechanical and metabolic roles in the body. Skeletal muscle is a major site for glucose disposal following a meal. Consequently, skeletal muscle plays an important role in postprandial blood glucose homeostasis. Over the past number of decades, research has demonstrated that insulin has an important role in vasodilating the vasculature in skeletal muscle in response to an insulin infusion (hyperinsulinaemic-euglycaemic clamp) or following the ingestion of a meal. This vascular action of insulin is pivotal for glucose disposal in skeletal muscle, as insulin-stimulated vasodilation increases the delivery of both glucose and insulin to the myocyte. Notably, in insulin-resistant states such as obesity and type 2 diabetes, this vascular response of insulin in skeletal muscle is significantly impaired. Whereas the majority of work in this field has focussed on the action of insulin alone on skeletal muscle microvascular blood flow and myocyte glucose metabolism, there is less understanding of how the consumption of a meal may affect skeletal muscle blood flow. This is in part due to complex variations in glucose and insulin dynamics that occurs postprandially-with changes in humoral concentrations of glucose, insulin, amino acids, gut and pancreatic peptides-compared to the hyperinsulinaemic-euglycaemic clamp. This review will address the emerging body of evidence to suggest that postprandial blood flow responses in skeletal muscle may be a function of the nutritional composition of a meal.

Advantages of an Anterior-Based Muscle-Sparing Approach in Transitioning From a Posterior Approach for Total Hip Arthroplasty: Minimizing the Learning Curve.

BACKGROUND: Enthusiasm for anterior-based approaches for total hip arthroplasty (THA) continues to increase but there is concern for increased complications during the learning curve period associated. This study aimed to investigate if there was a difference in perioperative variables, intraoperative and immediate postoperative complications, or patient-reported outcomes when transitioning from a mini-posterior approach (mPA) to an anterior-based muscle-sparing (ABMS) approach for THA. METHODS: Retrospective cohort study on the first 100 primary THA cases (n = 96 patients) of the senior author (August 2016 to August 2017) using the ABMS approach. These cases were compared to primary THA cases done the year prior (July 2015 to July 2016, n = 91 cases in 89 patients) using an mPA. Data were extracted and analyzed via gamma regression with robust standard errors and using generalized estimating equation regression. RESULTS: We found no difference in the estimated blood loss (P = .452) and surgical time (P = .564) between the cohorts. The ABMS cases had a slightly shorter length of stay (P = .001) with an adjusted mean length of stay of 1.53 days (95% confidence interval 1.4-1.6) compared to 1.85 days (95% confidence interval 1.8-1.9) in the mPA cases. There was no difference in the frequency of immediate postoperative complications (all, P > .05). There was no difference in the adjusted mean change in patient-reported outcomes (all P > .05). In the ABMS group, there was no difference in surgical time or physical function computerized adaptive test between the first 20 cases (reference) and each subsequent group of 20 cases (all P > .05). CONCLUSION: This study demonstrates no associated learning curve for an experienced senior surgeon when switching routine THA approach from mPA to ABMS. We advise careful interpretation of our results, as they may not apply to all surgeons and practices. LEVEL OF EVIDENCE: Level III Therapeutic Study: retrospective comparative study.

Oral glucose challenge impairs skeletal muscle microvascular blood flow in healthy people.

Skeletal muscle microvascular (capillary) blood flow increases in the postprandial state or during insulin infusion due to dilation of precapillary arterioles to augment glucose disposal. This effect occurs independently of changes in large artery function. However, acute hyperglycemia impairs vascular function, causes insulin to vasoconstrict precapillary arterioles, and causes muscle insulin resistance in vivo. We hypothesized that acute hyperglycemia impairs postprandial muscle microvascular perfusion, without disrupting normal large artery hemodynamics, in healthy humans. Fifteen healthy people (5 F/10 M) underwent an oral glucose challenge (OGC, 50 g glucose) and a mixed-meal challenge (MMC) on two separate occasions (randomized, crossover design). At 1 h, both challenges produced a comparable increase (6-fold) in plasma insulin levels. However, the OGC produced a 1.5-fold higher increase in blood glucose compared with the MMC 1 h postingestion. Forearm muscle microvascular blood volume and flow (contrast-enhanced ultrasound) were increased during the MMC (1.3- and 1.9-fold from baseline, respectively, P < 0.05 for both) but decreased during the OGC (0.7- and 0.6-fold from baseline, respectively, P < 0.05 for both) despite a similar hyperinsulinemia. Both challenges stimulated brachial artery flow (ultrasound) and heart rate to a similar extent, as well as yielding comparable decreases in diastolic blood pressure and total vascular resistance. Systolic blood pressure and aortic stiffness remained unaltered by either challenge. Independently of large artery hemodynamics, hyperglycemia impairs muscle microvascular blood flow, potentially limiting glucose disposal into skeletal muscle. The OGC reduced microvascular blood flow in muscle peripherally and therefore may underestimate the importance of skeletal muscle in postprandial glucose disposal.

Are the metabolic benefits of resistance training in type 2 diabetes linked to improvements in adipose tissue microvascular blood flow?

The microcirculation in adipose tissue is markedly impaired in type 2 diabetes (T2D). Resistance training (RT) often increases muscle mass and promotes a favorable metabolic profile in people with T2D, even in the absence of fat loss. Whether the metabolic benefits of RT in T2D are linked to improvements in adipose tissue microvascular blood flow is unknown. Eighteen sedentary people with T2D (7 women/11 men, 52 ± 7 yr) completed 6 wk of RT. Before and after RT, overnight-fasted participants had blood sampled for clinical chemistries (glucose, insulin, lipids, HbA1c, and proinflammatory markers) and underwent an oral glucose challenge (OGC; 50 g glucose × 2 h) and a DEXA scan to assess body composition. Adipose tissue microvascular blood volume and flow were assessed at rest and 1 h post-OGC using contrast-enhanced ultrasound. RT significantly reduced fasting blood glucose ( P = 0.006), HbA1c ( P = 0.007), 2-h glucose area under the time curve post-OGC ( P = 0.014), and homeostatic model assessment of insulin resistance ( P = 0.005). This was accompanied by a small reduction in total body fat ( P = 0.002), trunk fat ( P = 0.023), and fasting triglyceride levels ( P = 0.029). Lean mass ( P = 0.003), circulating TNF-α ( P = 0.006), and soluble VCAM-1 ( P < 0.001) increased post-RT. There were no significant changes in adipose tissue microvascular blood volume or flow following RT; however those who did have a higher baseline microvascular blood flow post-RT also had lower fasting triglyceride levels ( r = -0.476, P = 0.045). The anthropometric, glycemic, and insulin-sensitizing benefits of 6 wk of RT in people with T2D are not associated with an improvement in adipose tissue microvascular responses; however, there may be an adipose tissue microvascular-linked benefit to fasting triglyceride levels.

Regulation of microvascular flow and metabolism: An overview.

Skeletal muscle is an important site for insulin to regulate blood glucose levels. It is estimated that skeletal muscle is responsible for ~80% of insulin-mediated glucose disposal in the post-prandial period. The classical action of insulin to increase muscle glucose uptake involves insulin binding to insulin receptors on myocytes to stimulate glucose transporter 4 (GLUT 4) translocation to the cell surface membrane, enhancing glucose uptake. However, an additional role of insulin that is often under-appreciated is its action to increase muscle perfusion thereby improving insulin and glucose delivery to myocytes. Either of these responses (myocyte and/or vascular) may be impaired in insulin resistance, and both impairments are apparent in type 2 diabetes, resulting in diminished glucose disposal by muscle. The aim of this review is to report on the growing body of literature suggesting that insulin-mediated control of skeletal muscle perfusion is an important regulator of muscle glucose uptake and that impairment of microvascular insulin action has important physiological consequences early in the pathogenesis of insulin resistance. This work was discussed at the 2015 Australian Physiological Society Symposium "Physiological mechanisms controlling microvascular flow and muscle metabolism".

Muscle microvascular blood flow responses in insulin resistance and ageing.

Insulin resistance plays a key role in the development of type 2 diabetes. Skeletal muscle is the major storage site for glucose following a meal and as such has a key role in maintenance of blood glucose concentrations. Insulin resistance is characterised by impaired insulin-mediated glucose disposal in skeletal muscle. Multiple mechanisms can contribute to development of muscle insulin resistance and our research has demonstrated an important role for loss of microvascular function within skeletal muscle. We have shown that insulin can enhance blood flow to the microvasculature in muscle thus improving the access of glucose and insulin to the myocytes to augment glucose disposal. Obesity, insulin resistance and ageing are all associated with impaired microvascular responses to insulin in skeletal muscle. Impairments in insulin-mediated microvascular perfusion in muscle can directly cause insulin resistance, and this event can occur early in the aetiology of this condition. Understanding the mechanisms involved in the loss of microvascular function in muscle has the potential to identify novel treatment strategies to prevent or delay progression of insulin resistance and type 2 diabetes.

Late pleistocene Australian marsupial DNA clarifies the affinities of extinct megafaunal kangaroos and wallabies.

Understanding the evolution of Australia's extinct marsupial megafauna has been hindered by a relatively incomplete fossil record and convergent or highly specialized morphology, which confound phylogenetic analyses. Further, the harsh Australian climate and early date of most megafaunal extinctions (39-52 ka) means that the vast majority of fossil remains are unsuitable for ancient DNA analyses. Here, we apply cross-species DNA capture to fossils from relatively high latitude, high altitude caves in Tasmania. Using low-stringency hybridization and high-throughput sequencing, we were able to retrieve mitochondrial sequences from two extinct megafaunal macropodid species. The two specimens, Simosthenurus occidentalis (giant short-faced kangaroo) and Protemnodon anak (giant wallaby), have been radiocarbon dated to 46-50 and 40-45 ka, respectively. This is significantly older than any Australian fossil that has previously yielded DNA sequence information. Processing the raw sequence data from these samples posed a bioinformatic challenge due to the poor preservation of DNA. We explored several approaches in order to maximize the signal-to-noise ratio in retained sequencing reads. Our findings demonstrate the critical importance of adopting stringent processing criteria when distant outgroups are used as references for mapping highly fragmented DNA. Based on the most stringent nucleotide data sets (879 bp for S. occidentalis and 2,383 bp for P. anak), total-evidence phylogenetic analyses confirm that macropodids consist of three primary lineages: Sthenurines such as Simosthenurus (extinct short-faced kangaroos), the macropodines (all other wallabies and kangaroos), and the enigmatic living banded hare-wallaby Lagostrophus fasciatus (Lagostrophinae). Protemnodon emerges as a close relative of Macropus (large living kangaroos), a position not supported by recent morphological phylogenetic analyses.

Enhancement of insulin-mediated rat muscle glucose uptake and microvascular perfusion by 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside.

BACKGROUND: Insulin-induced microvascular recruitment is important for optimal muscle glucose uptake. 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR, an activator of AMP-activated protein kinase), can also induce microvascular recruitment, at doses that do not acutely activate glucose transport in rat muscle. Whether low doses of AICAR can augment physiologic insulin action is unknown. In the present study we used the euglycemic hyperinsulinemic clamp to assess whether insulin action is augmented by low dose AICAR. METHODS: Anesthetized rats were studied during saline infusion or euglycemic insulin (3 mU/kg/min) clamp for 2 h in the absence or presence of AICAR for the last hour (5 mg bolus followed by 3.75 mg/kg/min). Muscle glucose uptake (R'g) was determined radioisotopically with (14)C-2-deoxyglucose and muscle microvascular perfusion by contrast-enhanced ultrasound with microbubbles. RESULTS: AICAR did not affect blood glucose, or lower leg R'g, although it significantly (p < 0.05) increased blood lactate levels and augmented muscle microvascular blood volume via a nitric oxide synthase dependent pathway. Insulin increased femoral blood flow, whole body glucose infusion rate (GIR), R'g, hindleg glucose uptake, and microvascular blood volume. Addition of AICAR during insulin infusion increased lactate production, further increased R'g in Type IIA (fast twitch oxidative) and IIB (fast twitch glycolytic) fiber containing muscles, and hindleg glucose uptake, but decreased R'g in the Type I (slow twitch oxidative) fiber muscle. AICAR also decreased GIR due to inhibition of insulin-mediated suppression of hepatic glucose output. AICAR augmented insulin-mediated microvascular perfusion. CONCLUSIONS: AICAR, at levels that have no direct effect on muscle glucose uptake, augments insulin-mediated microvascular blood flow and glucose uptake in white fiber type muscles. Agents targeted to endothelial AMPK activation are promising insulin sensitizers, however, the decrease in GIR and the propensity to increase blood lactate cautions against AICAR as an acute insulin sensitizer.

A vascular mechanism for high-sodium-induced insulin resistance in rats.

AIMS/HYPOTHESIS: High sodium (HS) effects on hypertension are well established. Recent evidence implicates a relationship between HS intake and insulin resistance, even in the absence of hypertension. The aim of the current study was to determine whether loss of the vascular actions of insulin may be the driving factor linking HS intake to insulin resistance. METHODS: Sprague Dawley rats were fed a control (0.31% wt/wt NaCl) or HS (8.00% wt/wt NaCl) diet for 4 weeks and subjected to euglycaemic-hyperinsulinaemic clamp (10 mU min(-1) kg(-1)) or constant-flow pump-perfused hindlimb studies following an overnight fast. A separate group of HS rats was given quinapril during the dietary intervention and subjected to euglycaemic-hyperinsulinaemic clamp as above. RESULTS: HS intake had no effect on body weight or fat mass or on fasting glucose, insulin, endothelin-1 or NEFA concentrations. However, HS impaired whole body and skeletal muscle glucose uptake, in addition to a loss of insulin-stimulated microvascular recruitment. These effects were present despite enhanced insulin signalling (Akt) in both liver and skeletal muscle. Constant-flow pump-perfused hindlimb experiments revealed normal insulin-stimulated myocyte glucose uptake in HS-fed rats. Quinapril treatment restored insulin-mediated microvascular recruitment and muscle glucose uptake in vivo. CONCLUSIONS/INTERPRETATION: HS-induced insulin resistance is driven by impaired microvascular responsiveness to insulin, and is not due to metabolic or signalling defects within myocytes or liver. These results imply that reducing sodium intake may be important not only for management of hypertension but also for insulin resistance, and highlight the vasculature as a potential therapeutic target in the prevention of insulin resistance.

Does estrogen deficiency cause lacrimal gland inflammation and aqueous-deficient dry eye in mice?

Researchers have proposed that estrogen deficiency will lead to a Sjögren's syndrome (SjS)-like lacrimal gland inflammation, aqueous tear deficiency and dry eye. The purpose of this study was to determine whether this proposal is correct. Lacrimal glands were obtained from adult, age-matched wild type (WT) and aromatase knockout (ArKO) mice, in which estrogen synthesis is completely eliminated. Tissues were also obtained from autoimmune MRL/Mp-lpr/lpr (MRL/lpr) mice as inflammation controls. Tear volumes in WT and ArKO mice were measured and glands were processed for molecular biological and histological evaluation. Our results demonstrate that estrogen absence does not lead to a SjS-like inflammation in lacrimal tissue or to an aqueous-deficient dry eye. There was no upregulation of genes associated with inflammatory pathways in lacrimal glands of male or female ArKO mice. Such inflammatory activity was prominent in autoimmune MRL/lpr tissues. We also found no evidence of inflammation in lacrimal gland tissue sections of estrogen-deficient mice, and tear volumes of ArKO males were actually increased as compared to those WT controls. Interestingly, our study did show that estrogen absence influences the expression of thousands of lacrimal gland genes, and that this impact is sex- and genotype-specific. Our findings demonstrate that estrogen absence is not a risk factor for the development of SjS-like lacrimal gland inflammation or for aqueous-deficient dry eye in mice.

Local NOS inhibition impairs vascular and metabolic actions of insulin in rat hindleg muscle in vivo.

Insulin stimulates microvascular recruitment in skeletal muscle, and this vascular action augments muscle glucose disposal by ∼40%. The aim of the current study was to determine the contribution of local nitric oxide synthase (NOS) to the vascular actions of insulin in muscle. Hooded Wistar rats were infused with the NOS inhibitor N(ω)-nitro-L-arginine methylester (L-NAME, 10 µM) retrogradely via the epigastric artery in one leg during a systemic hyperinsulinemic-euglycemic clamp (3 mU·min(-1)·kg(-1) × 60 min) or saline infusion. Femoral artery blood flow, microvascular blood flow (assessed from 1-methylxanthine metabolism), and muscle glucose uptake (2-deoxyglucose uptake) were measured in both legs. Local L-NAME infusion did not have any systemic actions on blood pressure or heart rate. Local L-NAME blocked insulin-stimulated changes in femoral artery blood flow (84%, P < 0.05) and microvascular recruitment (98%, P < 0.05), and partially blocked insulin-mediated glucose uptake in muscle (reduced by 34%, P < 0.05). L-NAME alone did not have any metabolic effects in the hindleg. We conclude that insulin-mediated microvascular recruitment is dependent on local activation of NOS in muscle and that this action is important for insulin's metabolic actions.

Induction of Type 2 Diabetes in Mice to Understand Vascular Changes That Drive Diabetic Retinopathy.

Diabetic retinopathy is a common complication of type 2 diabetes. Research into this comorbidity is challenging due to the slow progression of pathological changes and the limited transgenic models available to study disease progression and mechanistic changes. Here, we describe a non-transgenic mouse model of accelerated type 2 diabetes using a high-fat diet in combination with streptozotocin delivered via osmotic mini pump. This model, when subjected to fluorescent gelatin vascular casting, can be used to study vascular changes in type 2 diabetic retinopathy.

Discovering the Secrets of Ancient Plants: Recovery of DNA from Museum and Archaeological Plant Specimens.

Plant DNA preserved in ancient specimens has recently gained importance as a tool in comparative genomics, allowing the investigation of evolutionary processes in plant genomes through time. However, recovering the genomic information contained in such specimens is challenging owing to the presence of secondary substances that limit DNA retrieval. In this chapter, we provide a DNA extraction protocol optimized for the recovery of DNA from degraded plant materials. The protocol is based on a commercially available DNA extraction kit that does not require handling of hazardous reagents.

Recovery of chloroplast genomes from medieval millet grains excavated from the Areni-1 cave in southern Armenia.

Panicum miliaceum L. was domesticated in northern China at least 7000 years ago and was subsequentially adopted in many areas throughout Eurasia. One such locale is Areni-1 an archaeological cave site in Southern Armenia, where vast quantities archaeobotanical material were well preserved via desiccation. The rich botanical material found at Areni-1 includes P. miliaceum grains that were identified morphologically and14C dated to the medieval period (873 ± 36 CE and 1118 ± 35 CE). To investigate the demographic and evolutionary history of the Areni-1 millet, we used ancient DNA extraction, hybridization capture enrichment, and high throughput sequencing to assemble three chloroplast genomes from the medieval grains and then compared these sequences to 50 modern P. miliaceum chloroplast genomes. Overall, the chloroplast genomes contained a low amount of diversity with domesticated accessions separated by a maximum of 5 SNPs and little inference on demography could be made. However, in phylogenies the chloroplast genomes separated into two clades, similar to what has been reported for nuclear DNA from P. miliaceum. The chloroplast genomes of two wild (undomesticated) accessions of P. miliaceum contained a relatively large number of variants, 11 SNPs, not found in the domesticated accessions. These results demonstrate that P. miliaceum grains from archaeological sites can preserve DNA for at least 1000 years and serve as a genetic resource to study the domestication of this cereal crop.

Adiponectin opposes endothelin-1-mediated vasoconstriction in the perfused rat hindlimb.

Recent studies have shown that adiponectin is able to increase nitric oxide (NO) production by the endothelium and relax preconstricted isolated aortic rings, suggesting that adiponectin may act as a vasodilator. Endothelin-1 (ET-1) is a potent vasoconstrictor, elevated levels of which are associated with obesity, type 2 diabetes, hypertension, and cardiovascular disease. We hypothesized that adiponectin has NO-dependent vascular actions opposing the vasoconstrictor actions of ET-1. We studied the vascular and metabolic effects of a physiological concentration of adiponectin (6.5 µg/ml) on hooded Wistar rats in the constant-flow pump-perfused rat hindlimb. Adiponectin alone had no observable vascular activity; however, adiponectin pretreatment and coinfusion inhibited the increase in perfusion pressure and associated metabolic stimulation caused by low-dose (1 nM) ET-1. Adiponectin was not able to oppose vasoconstriction when infusion was commenced after ET-1. This is in contrast to the NO donor sodium nitroprusside, which significantly reduced the pressure due to established ET-1 vasoconstriction, suggesting dissociation of the actions of adiponectin and NO. In addition, adiponectin had no effect on vasoconstriction caused by either high-dose (20 nM) ET-1 or low-dose (50 nM) norepinephrine. Our findings suggest that adiponectin has specific, apparently NO-independent, vascular activity to oppose the vasoconstrictor effects of ET-1. The hemodynamic actions of adiponectin may be an important aspect of its insulin-sensitizing ability by regulating access of insulin and glucose to myocytes. Imbalance in the relationship between adiponectin and ET-1 in obesity may contribute to the development of insulin resistance and cardiovascular disease.