Cigarette smoking is a risk factor for abdominal aortic aneurysms (AAAs), with studies suggesting a higher smoking-related AAA risk in women than men. We examined nicotine's effects on angiotensin II (AngII)-induced AAAs in male and female low-density lipoprotein receptor-deficient (Ldlr-/-) mice. Moreover, we defined effects of gonadectomy (GDX) of both sexes on nicotine-induced regulation of AAAs. Male and female Ldlr-/- mice (8-12 weeks of age) were infused with AngII with or without nicotine. Mice under- went sham or GDX surgeries prior to infusions of AngII and nicotine. In males, one or both testes were removed. AAA incidence, size, severity, and serum indices of nicotine metabolism were quantified. Effects of testosterone or estrogen on abdominal aortic smooth muscle cells (SMCs) were assessed. Nicotine increased aortic rupture in males, with modest effects in females. GDX reduced AAA incidence in male mice but had modest effects in females. Serum ratios of trans-3-hydroxycotinine to cotinine, an index of nicotine metabolism, were higher in females and increased by GDX in both sexes. Co-infusion of nicotine with AngII increased matrix metalloproteinase 2 (MMP2) mRNA in abdominal aortas of males, but not females. Similarly, testosterone increased MMP2 mRNA in male, but not female abdominal aortic SMCs. Testosterone reduced markers of a contractile SMC phenotype in SMCs from males, with no effects of estrogen in females. In conclusion, nicotine augments AngII-induced AAAs to a greater extent in males, with sex differences related to influences of sex hormones on nicotine metabolism, aortic MMP2 expression, and markers of a contractile SMC phenotype.

Serotonin (5-HT) has been implicated in cerebral aneurysm rupture, but it is unclear whether 5-HT plays a role in aortic aneurysm development and rupture, despite well known contractile effects of 5-HT through aortic 5-HT receptors. Abdominal aortic aneurysms (AAAs) induced by angiotensin II (AngII) infusion to mice exhibit periaortic inflammation and are prone to rupture. Periaortic fat (PAF), a potential source of 5-HT through tryptophan hydroxylase 1 (Tph1), has been implicated in AAA development. We quantified mRNA abundance of 5-HT receptors (Htr1b, Htr2a, Htr2b, Htr3a, and Htr7) and Tph1 in thoracic and abdominal aortas and surrounding PAF. Compared with other 5-HT receptors, we detected high levels of serotonin 3 receptor type a (Htr3a) mRNA in the abdominal aortas and abdominal PAF. Tph1 mRNA and 5-HT immunostaining were detected in aortas and PAF, with 5-HT levels higher in abdominal than thoracic PAF, and higher in epididymal white than interscapular brown fat. AngII infusion facilitated evoked [3H]5-HT release from thoracic PAF and modestly reduced 5-HT levels in thoracic PAF and brown fat. Based on a high level of Htr3a mRNA in abdominal aortas and PAF, we investigated the development of AngII-induced AAAs when serotonin 3 receptors were pharmacologically antagonized with tropisetron. Tropisetron abrogated abdominal aortic lumen diameters, aneurysm (distal thoracic aneurysm and AAA) incidence, maximal AAA diameters, and aortic weights of AngII-infused male mice. These findings indicate a novel role for serotonin 3 receptor in AAA development, with a potential clinically relevant contribution for PAF as a local source of 5-HT. SIGNIFICANCE STATEMENT: Aortic aneurysms are life-threatening vascular disorders with no effective therapeutics. This study identified antagonism of the serotonin 3 receptor as a potential therapeutic target to reduce the formation and severity of experimentally-induced aneurysms in the thoracic and abdominal aorta. Additionally, periaortic fat was identified as a potential site for serotonin production in the development of aortic aneurysms.

Opioid use disorder (OUD) is associated with nearly two-fold increased risk of cardiovascular disease, metabolic syndrome and diabetes. OUD by pregnant women is an understudied area related to the opioid epidemic leading to increased incidence of neonatal abstinence syndrome (NAS) and neonatal opioid withdrawal syndrome (NOWS). There is an interaction between the Renin-Angiotensin System (RAS) and the endogenous opioid system (EOS) commonly associated with the development of hypertension. This study tests the hypothesis that in utero opioid exposure induces hypertension and increases vascular reactivity to Angiotensin-II (AngII) in the adult offspring. Sprague Dawley dams were treated with escalating doses of a mu-opioid receptor agonist morphine (MOR, 5-20mg/kg/day, s.c) or saline (VEH). Weight gain was not different between MOR and VEH-exposed pregnant rats (F-MOR 100.2±9.5 vs F-VEH 95±10 g, n=4, p=NS). Additionally, litter size was not different (F-MOR13.75±0.2 vs F-VEH 12.25±1.8 pups, n=4, p=NS). However, MOR-exposed females exhibited low birth weight (F-MOR 5.6±0.2 vs F-VEH 7.2±0.6 g, n=20-22, p<0.05). Both MOR-exposed male and female offspring weighed less at weaning compared to VEH-exposed offspring (p<0.05). At 16 weeks on CHOW diet, only MOR-exposed female offspring continue to weigh significantly less than VEH-exposed offspring (F-MOR 264.5±9.7 vs F-VEH 306.5±14 g, n=14-16, p<0.05). Furthermore, both MOR-exposed male and female rats are hypertensive (M-MOR 127±0.84 vs M-VEH 110±1.45 mmHg, p<0.05; F-MOR 115.2±0.49 vs F-VEH 93.5±0.50 mmHg, p<0.05). MOR-exposed offspring displayed exacerbated acute pressor response to Angiotensin II (AngII, 0.01-1 ng/Kg, intraarterial), the effect was greater in MOR-exposed males than in females. Additionally, MOR-exposed females show impaired maximal relaxation in response to acetylcholine (Ach, 10-5 to 10-9 M, F-MOR 22.2±6 vs F-VEH 44.7±4 %, p<0.05) but not MOR-exposed males. However, only MOR-exposed males show exacerbated maximal AngII-induced vasoconstriction (M-MOR 49.4±4 vs M-VEH 35.8±3.9 %, p<0.05), which was blunted by an opioid receptor antagonist preincubation (naloxone, 1mM, 10 min, M-MOR=49.4±4 vs M-MOR+NAL 34.1±2.2 % max constriction, p<0.05; M-VEH=35.8±3.9 vs M-VEH+NAL=45.7±4 % max constriction, p=NS). At 16 weeks on high fat diet (HFD, 60% kcal from fat), male MOR-exposed offspring displayed weight gain in response to HFD (M-MOR 556.4±27 vs M-VEH 598.3±12.4 g, p=NS), while female MOR-exposed offspring were resistant to show diet-induced obesity (F-MOR 295.1±13 vs F-VEH 350.6±22 g, p<0.05). HFD impaired maximal relaxation in response to acetylcholine in VEH-exposed females (F-VEH 44.7±4 vs. F-VEH+HF 19±5 %, p<0.05) and MOR-exposed males relaxation (M-MOR 49.4±4 vs M-MOR+HF 31.2±3 %, p<0.05) but not VEH-exposed males or MOR-exposed females. Furthermore, circulating AngII was increased in MOR-exposed males (p<0.05 vs VEH). Maternal MOR exposure increases the risk of outcomes linked to cardiovascular and metabolic disease in adult offspring. The effects appeared to be mediated through a sex-specific mechanism in hypertension development involving potential crosstalk between AngII and the EOS.

Combined neprilysin (NEP) inhibition (sacubitril) and angiotensin type 1 receptor (AT1R) antagonism (valsartan) is used in the treatment of congestive heart failure and is gaining interest for other angiotensin II (AngII)-related cardiovascular diseases. In addition to heart failure, AngII promotes hypertension, atherosclerosis, and abdominal aortic aneurysms (AAAs). Similarly, NEP substrates or products have broad effects on the cardiovascular system. In this study, we examined NEP inhibition (with sacubitril) and AT1R antagonism (with valsartan) alone or in combination on AngII-induced hypertension, atherosclerosis, or AAAs in male low-density lipoprotein receptor-deficient mice. Preliminary studies assessed drug delivery via osmotic minipumps for simultaneous release of sacubitril and/or valsartan with AngII over 28 days. Mice were infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of sacubitril (1, 6, or 9 mg/kg per day), valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), or the combination thereof (1 and 0.3, or 9 or 0.5 mg/kg per day of sacubitril and valsartan, respectively). Plasma AngII and renin concentrations increased 4-fold at higher valsartan doses, indicative of removal of AngII negative feedback on renin. Sacubitril doubled plasma AngII concentrations at lower doses (1 mg/kg per day). Valsartan dose-dependently decreased systolic blood pressure, aortic atherosclerosis, and AAAs of AngII-infused mice, whereas sacubitril had no effect on atherosclerosis or AAAs but reduced blood pressure of AngII-infused mice. Combination therapy with sacubitril and valsartan did not provide additive benefits. These results suggest limited effects of combination therapy with NEP inhibition and AT1R antagonism against AngII-induced hypertension, atherosclerosis, or AAAs. SIGNIFICANCE STATEMENT: The combination of valsartan (angiotensin type 1 receptor antagonist) and sacubitril (neprilysin inhibitor) did not provide benefit above valsartan alone on AngII-induced hypertension, atherosclerosis, or abdominal aortic aneurysms in low-density lipoprotein receptor-deficient male mice. These results do not support this drug combination in therapy of these AngII-induced cardiovascular diseases.

Angiotensin converting enzyme 2 (ACE2) is an enzyme that limits activity of the renin-angiotensin system (RAS) and also serves as a receptor for the SARS-CoV-2 Spike (S) protein. Binding of S protein to ACE2 causes internalization which activates local RAS. ACE2 is on the X chromosome and its expression is regulated by sex hormones. In this study, we defined ACE2 mRNA abundance and examined effects of S protein on ACE2 activity and/or angiotensin II (AngII) levels in pivotal tissues (lung, adipose) from male and female mice. In lung, ACE2 mRNA abundance was reduced following gonadectomy (GDX) of male and female mice and was higher in XX than XY mice of the Four Core Genotypes (FCG). Reductions in lung ACE2 mRNA abundance by GDX occurred in XX, but not XY FCG female mice. Lung mRNA abundance of ADAM17 and TMPRSS2, enzymes that shed cell surface ACE2 and facilitate viral cell entry, was reduced by GDX in male but not female mice. For comparison, adipose ACE2 mRNA abundance was higher in female than male mice and higher in XX than XY FCG mice. Adipose ADAM17 mRNA abundance was increased by GDX of male and female mice. S protein reduced ACE2 activity in alveolar type II epithelial cells and 3T3-L1 adipocytes. Administration of S protein to male and female mice increased lung AngII levels and decreased adipose ACE2 activity in male but not female mice. These results demonstrate that sex differences in ACE2 expression levels may impact local RAS following S protein exposures.

Angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor and an enzyme of the renin-angiotensin system (RAS), is on the X chromosome and stimulated by estrogen. Male sex is a risk factor for SARS-CoV-2 severity. Previous investigators demonstrated that the SARs-CoV-2 Spike (S) protein decreases tissue ACE2 by protein internalization or shedding. This study defined sex differences in tissue ACE2 expression and their impact on SARS-CoV-2 S protein regulation of ACE2 activity and AngII levels. Male and female intact or gonadectomized (GDX) low density lipoprotein receptor deficient ( Ldlr -/- ) mice, and Four Core Genotype (FCG) male (XY or XX) or female (XX or XY) mice were fed a Western diet for 4 months. In lung, ACE2 mRNA abundance was similar in male and female mice and reduced by GDX (Male XY intact: 1.04 ± 0.15; Female XX intact: 1.13 ± 0.13; Male XY GDX: 0.11 ± 0.03; Female XX GDX: 0.18 ± 0.04 ΔΔCt; P<0.05). Lungs from XX mice had higher ACE2 mRNA abundance than XY mice regardless of gonadal sex (P<0.05), and GDX reduced ACE2 mRNA abundance in lungs of XX, but not XY females (XX Female GDX: 0.18 ± 0.04; XY Female GDX: 0.38 ± 0.09; P<0.05). In adipose, XX females had higher ACE2 mRNA abundance than XY males (XX female: 5.4 ± 0.7; XY male: 1.0 ± 0.1; P<0.05), regardless of gonadal sex (XY females: 3.3 ± 0.7; XX males: 1.5 ± 0.3; P<0.05). Male XY and female XX Ldlr -/- mice were administered vehicle or SARS-CoV-2 S protein (2 nmol/kg, ip, 3 doses) with tissue harvest six hours later. In lung, AngII levels were increased by S protein in male, but not female mice (Male, vehicle: 12.3 ± 2.3; Male, S protein: 33.6 ± 7.1; Female, vehicle: 16.1 ± 2.0; Female, S protein: 20.2 ± 1.3 pg/μg protein; P<0.05). In adipose, ACE2 activity was reduced by S protein in male, but not female mice (Male, vehicle: 63.6 ± 13.9; Male, S protein: 26.1 ± 1.9; Female, vehicle: 32.5 ± 1.9; Female, S protein: 25.1 ± 1.3 RFU/hr/mg tissue; P<0.05). SARS-CoV-2 S protein (35 nM) decreased ACE2 activity in type II lung alveolar cells (Vehicle: 2.0 x 10 4 ; S protein: 1.2 x 10 4 RFU/10 6 cells) and 3T3-L1 adipocytes (Vehicle: 2.1 x 10 4 ± 0.3 x 10 4 ; S protein: 1.1 x 10 4 ± 0.8 x 10 3 RFU/10 5 cells; P<0.05). Biologic sex regulation of ACE2 may protect females from SARS-CoV-2 S protein-mediated ACE2 reductions and activation of the local RAS.

Objective: Angiotensin II (AngII) actions at angiotensin type 1 receptors (AT1R) contribute to the development of atherosclerosis and abdominal aortic aneurysms (AAAs), two vascular diseases exhibi...

Objective: Abdominal aortic aneurysms (AAAs) exhibit sexual dimorphism with males demonstrating a 4-fold higher incidence compared to females. Our lab has shown that both sex hormones and sex chrom...

Background We have previously reported that female mice exposed to maternal separation and early weaning (MSEW), a model of early life stress, show exacerbated diet‐induced obesity associated with hypertension. The goal of this study was to test whether MSEW promotes angiotensin II–dependent hypertension via activation of the renin‐angiotensin system in adipose tissue. Methods and Results MSEW was achieved by daily separations from the dam and weaning at postnatal day 17, while normally reared controls were weaned at postnatal day 21. Female controls and MSEW weanlings were placed on a low‐fat diet (LF, 10% kcal from fat) or high‐fat diet (HF, 60% kcal from fat) for 20 weeks. MSEW did not change mean arterial pressure in LF–fed mice but increased it in HF–fed mice compared with controls (P<0.05). In MSEW mice fed a HF, angiotensin II concentration in plasma and adipose tissue was elevated compared with controls (P<0.05). In addition, angiotensinogen concentration was increased solely in adipose tissue from MSEW mice (P<0.05), while angiotensin‐converting enzyme protein expression and activity were similar between groups. Chronic enalapril treatment (2.5 mg/kg per day, drinking water, 7 days) reduced mean arterial pressure in both groups of mice fed a HF (P<0.05) and abolished the differences due to MSEW. Acute angiotensin II–induced increases in mean arterial pressure (10 μg/kg SC) were attenuated in untreated MSEW HF–fed mice compared to controls (P<0.05); however, this response was similar between groups in enalapril‐treated mice. Conclusions The upregulation of angiotensinogen and angiotensin II in adipose tissue could be an important mechanism by which female MSEW mice fed a HF develop hypertension.

Pseudomonas aeruginosa, a leading cause of sepsis, produces pyocyanin, a blue-pigmented virulence factor. Sepsis is associated with cachexia, but mechanisms are unknown and conventional nutrition approaches are not effective treatments. Pyocyanin has affinity for the aryl hydrocarbon receptor (AhR), which is expressed on adipocytes and regulates adipocyte differentiation. The purpose of this study was to define in vitro and in vivo effects of pyocyanin on adipocyte differentiation and body weight regulation as relates to septic cachexia. In 3T3-L1 preadipocytes, pyocyanin activated AhR and its downstream marker CYP1a1, and reduced differentiation. Administration of pyocyanin to male C57BL/6J mice acutely reduced body temperature with altered locomotion, but caused sustained weight loss. Chronic pyocyanin administration to male and female C57BL/6J mice resulted in sustained reductions in body weight and fat mass, with adipose-specific AhR activation. Pyocyanin-treated male mice had decreased energy expenditure and physical activity, and increased adipose explant lipolysis. In females, pyocyanin caused robust reductions in body weight, adipose-specific AhR activation, and increased expression of inflammatory cytokines in differentiated adipocytes. These results demonstrate that pyocyanin reduces adipocyte differentiation and decreases body weight and fat mass in male and female mice, suggesting that pyocyanin may play a role in septic cachexia.

Obesity increases the risk for hypertension in both sexes, but the prevalence of hypertension is lower in females than in males until menopause, despite a higher prevalence of obesity in females. We previously demonstrated that angiotensin-converting enzyme 2 (ACE2), which cleaves the vasoconstrictor, angiotensin II (AngII), to generate the vasodilator, angiotensin-(1-7) (Ang-(1-7)), contributes to sex differences in obesity-hypertension. ACE2 expression in adipose tissue was influenced by obesity in a sex-specific manner, with elevated ACE2 expression in obese female mice. Moreover, estrogen stimulated adipose ACE2 expression and reduced obesity-hypertension in females. In this study, we hypothesized that deficiency of adipocyte ACE2 contributes to obesity-hypertension of females. We generated a mouse model of adipocyte ACE2 deficiency. Male and female mice with adipocyte ACE2 deficiency or littermate controls were fed a low (LF) or a high fat (HF) diet for 16 weeks and blood pressure was quantified by radiotelemetry. HF-fed mice of each sex and genotype were challenged by an acute AngII injection, and blood pressure response was quantified. To translate these findings to humans, we performed a proof-of-principle study in obese transwomen in which systemic angiotensin peptides and blood pressure were quantified prior to and after 12 weeks of gender-affirming 17β-estradiol hormone therapy. Adipocyte ACE2 deficiency had no effect on the development of obesity in either sex. HF feeding increased systolic blood pressures (SBP) of wild-type male and female mice compared to LF-fed controls. Adipocyte ACE2 deficiency augmented obesity-induced elevations in SBP in females, but not in males. Obese female, but not obese male mice with adipocyte ACE2 deficiency, had an augmented SBP response to acute AngII challenge. In humans, plasma 17β-estradiol concentrations increased in obese transwomen administered 17β-estradiol and correlated positively with plasma Ang-(1-7)/AngII balance, and negatively to SBP after 12 weeks of 17β-estradiol administration. Adipocyte ACE2 protects female mice from obesity-hypertension, and reduces the blood pressure response to systemic AngII. In obese transwomen undergoing gender-affirming hormone therapy, 17β-estradiol administration may regulate blood pressure via the Ang-(1-7)/AngII balance.

Ellagic acid (EA) is a polyphenolic compound with antiviral activity against chikungunya, a rapidly spreading new tropical disease transmitted to humans by mosquitoes and now affecting millions worldwide. The most common symptoms of chikungunya virus infection are fever and joint pain. Other manifestations of infection can include encephalitis and an arthritic joint swelling with pain that may persist for months or years after the initial infection. The disease has recently spread to the U.S.A., with locally-transmitted cases of chikungunya virus reported in Florida. There is no approved vaccine to prevent or medicine to treat chikungunya virus infections. In this study, the Estimated Daily Intake (EDI) of EA from the food supply established using the National Health and Nutrition Examination Survey (NHANES) is used to set a maximum dose of an EA formulation for a high priority clinical trial.

Source(s) of Funding: The work described was supported in part by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1TR001998, and through an IRC grant from the College of Pharmacy. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. A complete and formatted version of this article is a v a i l a b l e a t  https://sites.google.com/a/contactincontext.org/asrg-public/research/publications/ADHD%20Device%20Drug%20

Objective: This study determined the toxicity and toxicokinetics of polydatin when administered via oral gavage to Sprague Dawley rats daily for 4 weeks. Background: Polydatin continues to be investigated as a potential therapy for a variety of human diseases. Methods: Male and female Crl:CD(SD) rats were assigned to five groups, and different doses (0, 300, 600, 1200, or 3000 mg/kg/day) of polydatin were administered via oral gavage once daily for 29 days at a dose volume of 10 mL/kg. Assessment of toxicity was based on mortality, clinical observations, food consumption, body weights, ophthalmic examinations, and clinical and anatomic pathology. Blood samples were collected for toxicokinetic evaluations. Results: All animals survived to the end of the study. Animals given polydatin at ≥600 mg/kg/day exhibited lower mean body weight (≤10% compared with control) and less gain in mean body weight, which correlated with decreased food consumption, compared to control rats. Several minor clinical chemistry findings were observed at ≥600 mg/kg/day, but they were all of small magnitude, and were not considered adverse or toxicologically important. Polydatin related macroscopic findings included one animal with an enlarged cecum in the 1200 mg/kg/day group and 6 of 20 animals with enlarged cecums or colons in the 3000 mg/kg/day group. These findings were considered test article-related, although no microscopic correlate was present. In males given 1200 or 3000 mg/kg/day and females given 3000 mg/kg/day microscopic findings in the kidneys included tubular dilatation, hyaline droplets in the tubule cells (males only), erosion/ulceration of the transitional epithelium, acute pelvic inflammation (males only), and chronic active pelvic inflammation with hyperplasia of the transitional epithelium (females only). No marked sex differences were observed in polydatin Cmax and AUC0-24 values. No accumulation of polydatin was observed after multiple dosing. Conclusion: The only adverse test article-related finding noted in one female given 3000 mg/kg/day was chronic active pelvic inflammation and transitional cell hyperplasia. Based on these results, the no observed adverse effect level (NOAEL) is 1200 mg/kg/day for females and 3000 mg/kg/day for males.

The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

The primary objectives of this study were to evaluate the treatment effect of D-tagatose on glycemic control, determined by a statistically significant decrease in hemoglobin A1c (HbA1c), and safety profile of D-tagatose compared to placebo. The secondary objectives were to evaluate the treatment effects on fasting blood glucose, insulin, lipid profiles, changes in BMI, and the proportion of subjects achieving HbA1c targets of <7%. Type 2 diabetic patients not taking any blood glucose lowering medications were administered either 15 g of D-tagatose dissolved in 125-250 ml of water three times a day or placebo with meals. Reduction in HbA1c was statistically significant compared to placebo at all post-baseline time points in the ITT population. Additionally, secondary endpoints were achieved in the ITT population with regard to LDL, total cholesterol, fasting blood glucose, and proportion of subjects achieving HbA1c targets of <7%. D-tagatose was unable to lower triglycerides or raise HDL compared to placebo. A subgroup LOCF analysis on the ITT US population showed a greater and statistically significant LS mean reduction in HbA1c in the D-tagatose group at all post-baseline visits. Based on these results it is concluded that in the ITT population D-tagatose is an effective single agent at treating many of the therapy targets of type 2 diabetes including lowering fasting blood glucose and HbA1c, and lowering of LDL and total cholesterol.

Bioluminescent labels can be especially useful for in vivo and live animal studies due to the negligible bioluminescence background in cells and most animals, and the non-toxicity of bioluminescent reporter systems. Significant thermal stability of bioluminescent labels is essential, however, due to the longitudinal nature and physiological temperature conditions of many bioluminescent-based studies. To improve the thermostability of the bioluminescent protein aequorin, we employed random and rational mutagenesis strategies to create two thermostable double mutants, S32T/E156V and M36I/E146K, and a particularly thermostable quadruple mutant, S32T/E156V/Q168R/L170I. The double aequorin mutants, S32T/E156V and M36I/E146K, retained 4 and 2.75 times more of their initial bioluminescence activity than wild-type aequorin during thermostability studies at 37 °C. Moreover, the quadruple aequorin mutant, S32T/E156V/Q168R/L170I, exhibited more thermostability at a variety of temperatures than either double mutant alone, producing the most thermostable aequorin mutant identified thus far.

The primary objective of this study was to evaluate the safety and the effect of D-tagatose on the glycemic control of subjects with type 2 diabetes as determined by HbA1c levels at the end of 6 months of therapy using the subject's own baseline HbA1c level as a comparator. The determination of the minimal dose required to cause a statistically significant reduction in HbA1c was of particular interest. Eight weeks after screening, the qualifying subjects were randomized to receive one of three doses of D-tagatose: 2.5 g TID, 5.0 g TID or 7.5 g TID. Blood levels of HbA1c, fasting blood glucose concentrations, plasma lipids, changes in body weight, changes in body mass index, and change in insulin levels were checked at each study visit and at the end of the study. Treatment success, as measured by the reduction of HbA1c, was greatest for the 7.5 g D-tagatose dose group, although the difference between the treatments was not statistically significant. For fasting glucose, only the 7.5 g dosage group exhibited reductions from baseline at the 3- and 6-month time points. Mean body weights reduced in a dose-response fashion, with the 5.0 g and the 7.5 g D-tagatose doses providing the greatest reductions. D-tagatose at dosages of 2.5 g, 5.0 g, and 7.5 g TID for six months were well tolerated by this subject population. D-tagatose at 5.0 g TID was the minimal dose required to reduce HbA1c. D-tagatose at 7.5 g TID provided the greatest effect in most measured efficacy parameters.

The proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and repair, including DNA mismatch repair (MMR), an essential mechanism that ensures replication fidelity. PCNA is required for MMR at both the initiation and re‐synthesis steps. Recent studies have shown that PCNA is phosphorylated at tyrosine 211 (Y211) by epidermal growth factor receptor (EGFR), whose overexpression is associated with during tumor progression. We therefore hypothesize that the EGFR tumor‐promotion function is through PCNA Y211 phosphorylation by altering the MMR function. To test this hypothesis, we directly examined the influence of PCNA Y211 phosphorylation on MMR activity using a functional in vitro repair assay. We demonstrate here that nuclear extracts derived from tumor cells with high levels of phosphorylated PCNA are defective in MMR, and that the deficiency can be restored by purified non‐phosphorylated PCNA, suggesting that PCNA Y211 phosphorylation inhibits MMR. The inhibition was due to the inability of the phosphorylated PCNA to interact with mismatch recognition proteins MutSα and MutSβ. We also show that the PCNA‐phosphorylated nuclear extracts have a delayed DNA resynthesis during MMR, which is coupled with nucleotide misincorporations, leading to an elevated mutation frequency. Our study therefore demonstrates that a posttranslational modification promotes genome instability and tumor progression by negatively regulating the MMR function and DNA synthesis. Thus, the work provides a potential new biomarker for cancer progression.