Frailty increases the long-term risk for fall and fracture-related hospitalizations and all-cause mortality in community-dwelling older women.

Frailty is associated with declines in physiological capacity across sensory, neurological, and musculoskeletal systems. An underlying assumption is that the frailer an individual, the more likely they are to experience falls and fractures. We examined whether grades of frailty can assess the long-term risk of hospitalized falls, fractures, and all-cause mortality in 1261 community-dwelling older women (mean age [SD] of 75.1 [2.7] yr) over 14.5 yr. Frailty was operationalized using a frailty index (FI) of cumulative deficits from 33 variables across multiple health domains (physical, mental, comorbidities) at baseline. The total score across these variables was summed and divided by 33 to obtain the FI. Participants were graded as fit (FI ≤ 0.12), mildly frail (FI > 0.12-0.24), moderately frail (FI > 0.24-0.36), or severely frail (FI > 0.36). Fall-related (n = 498), any fracture-related (n = 347), and hip fracture-related hospitalizations (n = 137) and deaths (n = 482) were obtained from linked health records. Associations between FI grades and clinical outcomes were analyzed using multivariable-adjusted Cox-proportional hazard models including age, treatment (calcium/placebo), BMI, smoking history, socioeconomic status, plasma vitamin D (25OHD) status plus season obtained, physical activity, self-reported prevalent falls in the last 3 mo, and self-reported fractures since the age of 50 yr. At baseline, 713 (56.5%), 350 (27.8%), 163 (12.9%), and 35 (2.8%) of women were classified as fit, mildly frail, moderately frail, and severely frail, respectively. Women with mild, moderate, and severe frailty had significantly higher hazards (all P < .05) for a fall-related (46%, 104%, 168%), any fracture-related (88% for moderate, 193% for severe frailty), hip fracture-related hospitalizations (93%, 127%, 129%), and all-cause mortality (47%, 126%, 242%). The FI identified community-dwelling older women at risk for the most serious falls and fractures and may be incorporated into risk assessment tools to identify individuals with poorer clinical prognosis.

Frailty is often linked to decline in the sensory, neurological, and musculoskeletal systems. Frailty can be identified and graded using a frailty index (FI). In a cohort of 1261 community-dwelling older women with an average age of 75 yr, this study found that increasing grades of frailty fit, (mildly, moderately and severely frail) were associated with higher long-term risk for a fall and/or fracture (including hip fractures) that required hospitalization. Increasing grades of frailty were also linked with greater risk of death, especially due to cardiovascular causes. In conclusion, the FI may be used to identify community-dwelling older women with a high long-term risk for the most serious falls and fractures, as well as individuals with a poorer prognosis.

Undercarboxylated osteocalcin and ibandronate combination ameliorates hindlimb immobilization-induced muscle wasting.

Immobilization leads to muscle wasting and insulin resistance, particularly during ageing. It has been suggested that undercarboxylated osteocalcin (ucOC) improves muscle mass and glucose metabolism. Bisphosphonates, an anti-osteoporosis treatment, might protect muscle wasting independent of ucOC. We hypothesize that the combination of ucOC and ibandronate (IBN) treatments has superior protective effects against immobilization-induced muscle wasting and insulin resistance than either treatment alone. C57BL/6J mice were hindlimb-immobilized for two weeks, with injections of vehicle, ucOC (90 ng/g daily) and/or IBN (2 µg/g weekly). Insulin/oral glucose tolerance tests (ITT/OGTT) were performed. Immediately after immobilization, muscles (extensor digitorum longus (EDL), soleus, tibialis anterior, gastrocnemius and quadriceps) were isolated and measured for muscle mass. Insulin-stimulated glucose uptake (EDL and soleus) was examined. Phosphorylation/expression of proteins in anabolic/catabolic pathways were examined in quadriceps. Primary human myotubes derived from older adult muscle biopsies were treated with ucOC and/or IBN, then signalling proteins were analysed. Combined treatment, but not individual treatments, significantly increased the muscle weight/body weight ratio in immobilized soleus (31.7%; P = 0.013) and quadriceps (20.0%; P = 0.0008) muscles, concomitant with elevated p-Akt (S473)/Akt ratio (P = 0.0047). Combined treatment also enhanced whole-body glucose tolerance (16.6%; P = 0.0011). In human myotubes, combined treatment stimulated greater activation of ERK1/2 (P = 0.0067 and 0.0072) and mTOR (P = 0.036), and led to a lesser expression of Fbx32 (P = 0.049) and MuRF1 (P = 0.048) than individual treatments. These findings suggest a potential therapeutic role for the ucOC and bisphosphonates combination in protecting against muscle wasting induced by immobilization and ageing. KEY POINTS: It has been suggested that undercarboxylated osteocalcin (ucOC) improves muscle mass and glucose metabolism. Bisphosphonates, an anti-osteoporosis treatment, might protect against muscle wasting independent of ucOC. The combination treatment of ucOC and ibandronate was shown to exert a greater therapeutic effect against immobilization-induced muscle wasting, and led to greater activation of anabolic pathway and less expression of catabolic signalling proteins in myotubes derived from older adults, compared with individual treatments. The combination treatment was found to improve whole-body glucose tolerance. Our findings suggest a potential therapeutic role for the ucOC and bisphosphonates combination in protecting against muscle wasting induced by immobilization and ageing.

Wild Bee Exposure to Pesticides in Conservation Grasslands Increases along an Agricultural Gradient: A Tale of Two Sample Types.

Conservation efforts have been implemented in agroecosystems to enhance pollinator diversity by creating grassland habitat, but little is known about the exposure of bees to pesticides while foraging in these grassland fields. Pesticide exposure was assessed in 24 conservation grassland fields along an agricultural gradient at two time points (July and August) using silicone band passive samplers (nonlethal) and bee tissues (lethal). Overall, 46 pesticides were detected including 9 herbicides, 19 insecticides, 17 fungicides, and a plant growth regulator. For the bands, there were more frequent/higher concentrations of herbicides in July (maximum: 1600 ng/band in July; 570 ng/band in August), while insecticides and fungicides had more frequent/higher concentrations in August (maximum: 110 and 65 ng/band in July; 1500 and 1700 ng/band in August). Pesticide concentrations in bands increased 16% with every 10% increase in cultivated crops. The bee tissues showed no difference in detection frequency, and concentrations were similar among months; maximum concentrations of herbicides, insecticides, and fungicides in July and August were 17, 27, and 180 and 19, 120, and 170 ng/g, respectively. Pesticide residues in bands and bee tissues did not always show the same patterns; of the 20 compounds observed in both media, six (primarily fungicides) showed a detection-concentration relationship between the two media. Together, the band and bee residue data can provide a more complete understanding of pesticide exposure and accumulation in conserved grasslands.

NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP.

Miro2 and Miro1 are mitochondrial-associated proteins critical for regulating mitochondrial movement within the cell. Both Miro1 and Miro2 have roles in promoting neuron function, but recently Miro2 has been shown to have additional roles in response to nutrient starvation in tumor cells. Miro1 and 2 consist of two small GTPase domains flanking a pair of EF-hands. The N-terminal GTPase (nGTPase) domain is responsible for initiating mitochondrial trafficking and interactions with GCN1 in prostate cancer. The crystal structure of Miro1 nGTPase bound to GTP has been solved. However, no structural data is available for the nGTPase domain of Miro2. To better understand the similarities and differences in the functions of Miro1 and Miro2, we have initiated structural studies of Miro2. Here we report the backbone NMR chemical shift assignments of a 22 KDa construct of the nGTPase domain of Miro2 bound to GTP that includes residues 1-180 of the full-length protein. We affirm that the overall secondary structure of this complex closely resembles that of Miro1 nGTPase bound to GTP. Minor variations in the overall structures can be attributed to crystal packing interactions in the structure of Miro1. These NMR studies will form the foundation for future work identifying the specific interaction sites between Miro2 and its cellular binding partners.

DPP4 inhibition mitigates ANG II-mediated kidney immune activation and injury in male mice.

Recent evidence suggests that dipeptidyl peptidase-4 (DPP4) inhibition with saxagliptin (Saxa) is renoprotective under comorbid conditions associated with activation of the renin-angiotensin-aldosterone system (RAAS), such as diabetes, obesity, and hypertension, which confer a high cardiovascular risk. Immune system activation is now recognized as a contributor to RAAS-mediated tissue injury, and, importantly, immunomodulatory effects of DPP4 have been reported. Accordingly, we examined the hypothesis that DPP4 inhibition with Saxa attenuates angiotensin II (ANG II)-induced kidney injury and albuminuria via attenuation of immune activation in the kidney. To this end, male mice were infused with either vehicle or ANG II (1,000 ng/kg/min, s.c.) for 3 wk and received either placebo or Saxa (10 mg/kg/day, p.o.) during the final 2 wk. ANG II infusion increased kidney, but not plasma, DPP4 activity in vivo as well as DPP4 activity in cultured proximal tubule cells. The latter was prevented by angiotensin receptor blockade with olmesartan. Further, ANG II induced hypertension and kidney injury characterized by mesangial expansion, mitochondrial damage, reduced brush border megalin expression, and albuminuria. Saxa inhibited DPP4 activity ∼50% in vivo and attenuated ANG II-mediated kidney injury, independent of blood pressure. Further mechanistic experiments revealed mitigation by Saxa of proinflammatory and profibrotic mediators activated by ANG II in the kidney, including CD8+ T cells, resident macrophages (CD11bhiF4/80loLy6C-), and neutrophils. In addition, Saxa improved ANG II suppressed anti-inflammatory regulatory T cell and T helper 2 lymphocyte activity. Taken together, these results demonstrate, for the first time, blood pressure-independent involvement of renal DPP4 activation contributing to RAAS-dependent kidney injury and immune activation.NEW & NOTEWORTHY This work highlights the role of dipeptidyl peptidase-4 (DPP4) in promoting ANG II-mediated kidney inflammation and injury. Specifically, ANG II infusion in mice led to increases in blood pressure and kidney DPP4 activity, which then led to activation of CD8+ T cells, Ly6C- macrophages, and neutrophils and suppression of anti-inflammatory T helper 2 lymphocytes and regulatory T cells. Collectively, this led to kidney injury, characterized by mesangial expansion, mitochondrial damage, and albuminuria, which were mitigated by DPP4 inhibition independent of blood pressure reduction.

Proximal Cysteines that Enhance Lysine N-Acetylation of Cytosolic Proteins in Mice Are Less Conserved in Longer-Living Species.

Acetyl-coenzyme A (CoA) is an abundant metabolite that can also alter protein function through non-enzymatic N-acetylation of protein lysines. This N-acetylation is greatly enhanced in vitro if an adjacent cysteine undergoes initial S-acetylation, as this can lead to S→N transfer of the acetyl moiety. Here, using modeled mouse structures of 619 proteins N-acetylated in mouse liver, we show lysine N-acetylation is greater in vivo if a cysteine is within ∼10 Å. Extension to the genomes of 52 other mammalian and bird species shows pairs of proximal cysteine and N-acetylated lysines are less conserved, implying most N-acetylation is detrimental. Supporting this, there is less conservation of cytosolic pairs of proximal cysteine and N-acetylated lysines in species with longer lifespans. As acetyl-CoA levels are linked to nutrient supply, these findings suggest how dietary restriction could extend lifespan and how pathologies resulting from dietary excess may occur.

The Causes and Consequences of Nonenzymatic Protein Acylation.

Thousands of protein acyl modification sites have now been identified in vivo. However, at most sites the acylation stoichiometry is low, making functional enzyme-driven regulation in the majority of cases unlikely. As unmediated acylation can occur on the surface of proteins when acyl-CoA thioesters react with nucleophilic cysteine and lysine residues, slower nonenzymatic processes likely underlie most protein acylation. Here, we review how nonenzymatic acylation of nucleophilic lysine and cysteine residues occurs; the factors that enhance acylation at particular sites; and the strategies that have evolved to limit protein acylation. We conclude that protein acylation is an unavoidable consequence of the central role of reactive thioesters in metabolism. Finally, we propose a hypothesis for why low-stoichiometry protein acylation is selected against by evolution and how it might contribute to degenerative processes such as aging.

Effects of Dietary Inorganic Nitrate Supplementation on Exercise Performance in Patients With Heart Failure: Protocol for a Randomized, Placebo-Controlled, Cross-Over Trial.

BACKGROUND: Chronic heart failure is characterized by an inability of the heart to pump enough blood to meet the demands of the body, resulting in the hallmark symptom of exercise intolerance. Chronic underperfusion of the peripheral tissues and impaired nitric oxide bioavailability have been implicated as contributors to the decrease in exercise capacity in these patients. nitric oxide bioavailability has been identified as an important mediator of exercise tolerance in healthy individuals, but there are limited studies examining the effects in patients with chronic heart failure. OBJECTIVE: The proposed trial is designed to determine the effects of chronic inorganic nitrate supplementation on exercise tolerance in both patients with heart failure preserved ejection fraction (HFpEF) and heart failure reduced ejection fraction (HFrEF) and to determine whether there are any differential responses between the 2 cohorts. A secondary objective is to provide mechanistic insights into the 2 heart failure groups' exercise responses to the nitrate supplementation. METHODS: Patients with chronic heart failure (15=HFpEF and 15=HFrEF) aged 40 to 85 years will be recruited. Following an initial screen cardiopulmonary exercise test, participants will be randomly allocated in a double-blind fashion to consume either a nitrate-rich beetroot juice (16 mmol nitrate/day) or a nitrate-depleted placebo (for 5 days). Participants will continue daily dosing until the completion of the 4 testing visits (maximal cardiopulmonary exercise test, submaximal exercise test with echocardiography, vascular function assessment, and vastus lateralis muscle biopsy). There will then be a 2-week washout period after which the participants will cross over to the other treatment and complete the same 4 testing visits. RESULTS: This study is funded by National Heart Foundation of Australia and Victoria University. Enrolment has commenced and the data collection is expected to be completed in mid 2018. The initial results are expected to be submitted for publication by the end of 2018. CONCLUSIONS: If inorganic nitrate supplementation can improve exercise tolerance in patients with chronic heart failure, it has the potential to aid in further refining the treatment of patients in this population. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12615000906550; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=368912 (Archived by WebCite at http://www.webcitation.org/6xymLMiFK).

Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Ameliorates Angiotensin II-Induced Cardiac Diastolic Dysfunction in Male Mice.

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.

Dual-activity PI3K-BRD4 inhibitor for the orthogonal inhibition of MYC to block tumor growth and metastasis.

MYC is a major cancer driver but is documented to be a difficult therapeutic target itself. Here, we report on the biological activity, the structural basis, and therapeutic effects of the family of multitargeted compounds that simultaneously disrupt functions of two critical MYC-mediating factors through inhibiting the acetyllysine binding of BRD4 and the kinase activity of PI3K. We show that the dual-action inhibitor impairs PI3K/BRD4 signaling in vitro and in vivo and affords maximal MYC down-regulation. The concomitant inhibition of PI3K and BRD4 blocks MYC expression and activation, promotes MYC degradation, and markedly inhibits cancer cell growth and metastasis. Collectively, our findings suggest that the dual-activity inhibitor represents a highly promising lead compound for the development of novel anticancer therapeutics.

Reliable and High Efficiency Extraction of Kidney Immune Cells.

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

A non-human primate model of radiation-induced cachexia.

Cachexia, or muscle wasting, is a serious health threat to victims of radiological accidents or patients receiving radiotherapy. Here, we propose a non-human primate (NHP) radiation-induced cachexia model based on clinical and molecular pathology findings. NHP exposed to potentially lethal partial-body irradiation developed symptoms of cachexia such as body weight loss in a time- and dose-dependent manner. Severe body weight loss as high as 20-25% was observed which was refractory to nutritional intervention. Radiographic imaging indicated that cachectic NHP lost as much as 50% of skeletal muscle. Histological analysis of muscle tissues showed abnormalities such as presence of central nuclei, inflammation, fatty replacement of skeletal muscle, and muscle fiber degeneration. Biochemical parameters such as hemoglobin and albumin levels decreased after radiation exposure. Levels of FBXO32 (Atrogin-1), ActRIIB and myostatin were significantly changed in the irradiated cachectic NHP compared to the non-irradiated NHP. Our data suggest NHP that have been exposed to high dose radiation manifest cachexia-like symptoms in a time- and dose-dependent manner. This model provides a unique opportunity to study the mechanism of radiation-induced cachexia and will aid in efficacy studies of mitigators of this disease.

Increased Expression of Connective Tissue Growth Factor (CTGF) in Multiple Organs After Exposure of Non-Human Primates (NHP) to Lethal Doses of Radiation.

Exposure to sufficiently high doses of ionizing radiation is known to cause fibrosis in many different organs and tissues. Connective tissue growth factor (CTGF/CCN2), a member of the CCN family of matricellular proteins, plays an important role in the development of fibrosis in multiple organs. The aim of the present study was to quantify the gene and protein expression of CTGF in a variety of organs from non-human primates (NHP) that were previously exposed to potentially lethal doses of radiation. Tissues from non-irradiated NHP and NHP exposed to whole thoracic lung irradiation (WTLI) or partial-body irradiation with 5% bone marrow sparing (PBI/BM5) were examined by real-time quantitative reverse transcription PCR, western blot, and immunohistochemistry. Expression of CTGF was elevated in the lung tissues of NHP exposed to WTLI relative to the lung tissues of the non-irradiated NHP. Increased expression of CTGF was also observed in multiple organs from NHP exposed to PBI/BM5 compared to non-irradiated NHP; these included the lung, kidney, spleen, thymus, and liver. These irradiated organs also exhibited histological evidence of increased collagen deposition compared to the control tissues. There was significant correlation of CTGF expression with collagen deposition in the lung and spleen of NHP exposed to PBI/BM5. Significant correlations were observed between spleen and multiple organs on CTGF expression and collagen deposition, respectively, suggesting possible crosstalk between spleen and other organs. These data suggest that CTGF levels are increased in multiple organs after radiation exposure and that inflammatory cell infiltration may contribute to the elevated levels of CTGF in multiple organs.

The Effect of Radiation Dose and Variation in Neupogen® Initiation Schedule on the Mitigation of Myelosuppression during the Concomitant GI-ARS and H-ARS in a Nonhuman Primate Model of High-dose Exposure with Marrow Sparing.

A nonhuman primate (NHP) model of acute high-dose, partial-body irradiation with 5% bone marrow (PBI/BM5) sparing was used to assess the effect of Neupogen® [granulocyte colony stimulating factor (G-CSF)] to mitigate the associated myelosuppression when administered at an increasing interval between exposure and initiation of treatment. A secondary objective was to assess the effect of Neupogen® on the mortality or morbidity of the hematopoietic (H)- acute radiation syndrome (ARS) and concurrent acute gastrointestinal radiation syndrome (GI-ARS). NHP were exposed to 10.0 or 11.0 Gy with 6 MV LINAC-derived photons at approximately 0.80 Gy min. All NHP received medical management. NHP were dosed daily with control article (5% dextrose in water) initiated on day 1 post-exposure or Neupogen® (10 µg kg) initiated on day 1, day 3, or day 5 until recovery [absolute neutrophil count (ANC) ≥ 1,000 cells µL for three consecutive days]. Mortality in both the 10.0 Gy and 11.0 Gy cohorts suggested that early administration of Neupogen® at day 1 post exposure may affect acute GI-ARS mortality, while Neupogen® appeared to mitigate mortality due to the H-ARS. However, the study was not powered to detect statistically significant differences in survival. The ability of Neupogen® to stimulate granulopoiesis was assessed by evaluating key parameters for ANC recovery: the depth of nadir, duration of neutropenia (ANC < 500 cells µL) and recovery time to ANC ≥ 1,000 cells µL. Following 10.0 Gy PBI/BM5, the mean duration of neutropenia was 11.6 d in the control cohort vs. 3.5 d and 4.6 d in the day 1 and day 3 Neupogen® cohorts, respectively. The respective ANC nadirs were 94 cells µL, 220 cells µL, and 243 cells µL for the control and day 1 and day 3 Neupogen® cohorts. Following 11.0 Gy PBI/BM5, the duration of neutropenia was 10.9 d in the control cohort vs. 2.8 d, 3.8 d, and 4.5 d in the day 1, day 3, and day 5 Neupogen® cohorts, respectively. The respective ANC nadirs for the control and day 1, day 3, and day 5 Neupogen® cohorts were 131 cells µL, 292 cells µL, 236 cells µL, and 217 cells µL, respectively. Therefore, the acceleration of granulopoiesis by Neupogen® in this model is independent of the time interval between radiation exposure and treatment initiation up to 5 d post-exposure. The PBI/BM5 model can be used to assess medical countermeasure efficacy in the context of the concurrent GI- and H-ARS.

A MALDI-MSI Approach to the Characterization of Radiation-Induced Lung Injury and Medical Countermeasure Development.

Radiation-induced lung injury is highly complex and characterized by multiple pathologies, which occur over time and sporadically throughout the lung. This complexity makes biomarker investigations and medical countermeasure screenings challenging. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has the ability to resolve differences spatially in molecular profiles within the lung following radiation exposure and can aid in biomarker identification and pharmaceutical efficacy investigations. MALDI-MSI was applied to the investigation of a whole-thorax lung irradiation model in non-human primates (NHP) for lipidomic analysis and medical countermeasure distribution.

Pegfilgrastim Improves Survival of Lethally Irradiated Nonhuman Primates.

Leukocyte growth factors (LGF), such as filgrastim, pegfilgrastim and sargramostim, have been used to mitigate the hematologic symptoms of acute radiation syndrome (ARS) after radiation accidents. Although these pharmaceuticals are currently approved for treatment of chemotherapy-induced myelosuppression, such approval has not been granted for myelosuppression resulting from acute radiation exposure. Regulatory approval of drugs used to treat radiological or nuclear exposure injuries requires their development and testing in accordance with the Animal Efficacy Rule, set forth by the U.S. Food and Drug Administration. To date, filgrastim is the only LGF that has undergone efficacy assessment conducted under the Animal Efficacy Rule. To confirm the efficacy of another LGF with a shorter dosing regimen compared to filgrastim, we evaluated the use of pegfilgrastim (Neulasta(®)) in a lethal nonhuman primate (NHP) model of hematopoietic acute radiation syndrome (H-ARS). Rhesus macaques were exposed to 7.50 Gy total-body irradiation (the LD(50/60)), delivered at 0.80 Gy/min using linear accelerator 6 MV photons. Pegfilgrastim (300 µg/kg, n = 23) or 5% dextrose in water (n = 23) was administered on day 1 and 8 postirradiation and all animals received medical management. Hematologic and physiologic parameters were evaluated for 60 days postirradiation. The primary, clinically relevant end point was survival to day 60; secondary end points included hematologic-related parameters. Pegfilgrastim significantly (P = 0.0014) increased 60 day survival to 91.3% (21/23) from 47.8% (11/23) in the control. Relative to the controls, pegfilgrastim also significantly: 1. decreased the median duration of neutropenia and thrombocytopenia; 2. improved the median time to recovery of absolute neutrophil count (ANC) ≥500/µL, ANC ≥1,000/µL and platelet (PLT) count ≥20,000/µL; 3. increased the mean ANC at nadir; and 4. decreased the incidence of Gram-negative bacteremia. These data demonstrate that pegfilgrastim is an additional medical countermeasure capable of increasing survival and neutrophil-related parameters when administered in an abbreviated schedule to a NHP model of lethal H-ARS.

Copper-induced changes in intracellular thiols in two marine diatoms: Phaeodactylum tricornutum and Ceratoneis closterium.

Phytochelatins and glutathione (reduced (GSH) and oxidised (GSSG)) are important intracellular ligands involved in metal sequestration and detoxification in algae. Intracellular ratios of GSH:GSSG are sensitive indicators of metal stress in algae, and like phytochelatin production are influenced by metal speciation, concentration, exposure time and the biological species. This study investigated the effect of copper exposure on phytochelatin and glutathione content in two marine diatoms Phaeodactylum tricornutum and Ceratoneis closterium at various time intervals between 0.5 and 72h. Liberation of cellular glutathione and phytochelatins was optimised using freeze/thaw cycles and chemical extraction, respectively. Extracted phytochelatins were derivatised (by fluorescent tagging of thiol compounds), separated and quantified using HPLC with fluorescence detection. Glutathione ratios were determined using a commercially available kit, which uses the enzyme glutathione reductase to measure total and oxidised glutathione. Despite similarities in size and shape between the two diatoms, differences in internalised copper, phytochelatin production (both chain length and quantity) and reduced glutathione concentrations were observed. P. tricornutum maintained reduced glutathione at between 58 and 80% of total glutathione levels at all time points, which would indicate low cellular stress. In C. closterium reduced glutathione constituted <10% of total glutathione after 48h. P. tricornutum also produced more phytochelatins and phytochelatins of longer chain length than C. closterium despite the latter species internalising significantly more copper.

The ability of filgrastim to mitigate mortality following LD50/60 total-body irradiation is administration time-dependent.

The identification of the optimal administration schedule for an effective medical countermeasure is critical for the effective treatment of individuals exposed to potentially lethal doses of radiation. The efficacy of filgrastim (Neupogen®), a potential medical countermeasure, to improve survival when initiated at 48 h following total body irradiation in a non-human primate model of the hematopoietic syndrome of the acute radiation syndrome was investigated. Animals were exposed to total body irradiation, antero-posterior exposure, total midline tissue dose of 7.5 Gy, (target lethal dose 50/60) delivered at 0.80 Gy min, using linear accelerator-derived 6 MV photons. All animals were administered medical management. Following irradiation on day 0, filgrastim (10 µg kg d) or the control (5% dextrose in water) was administered subcutaneously daily through effect (absolute neutrophil count ≥ 1,000 cells µL for three consecutive days). The study (n = 80) was powered to demonstrate a 25% improvement in survival following the administration of filgrastim or control beginning at 48 ± 4 h post-irradiation. Survival analysis was conducted on the intention-to-treat population using a two-tailed null hypothesis at a 5% significance level. Filgrastim, initiated 48 h after irradiation, did not improve survival (2.5% increase, p = 0.8230). These data demonstrate that efficacy of a countermeasure to mitigate lethality in the hematopoietic syndrome of the acute radiation syndrome can be dependent on the interval between irradiation and administration of the medical countermeasure.

Conference scene: 2nd cancer epigenetics conference.

The GTC Cancer Summit: Novel Approaches to Drug Discovery was divided into two parallel tracks: the 2nd Cancer Epigenetics Conference, and the Protein Kinases and Drug Design Conference. The 2nd Cancer Epigenetics Conference focused on exciting changes in drug discovery that include an unprecedented private and public collaboration on drug discovery in epigenetics through the Structural Genomics Consortium (SGC), which has led to several major breakthroughs including: the development of small-molecule inhibitors that interfere with protein interactions, especially bromodomain-containing protein acetylation readers; the indirect but successful targeting of the elusive MYC oncogene; and the identification of epigenetic drugs that are disease-specific. Also reported were the development of clinically useful DNA methylation assays; cell, peptide and protein arrays for testing antibody- and protein-binding specificity; and tools for chromatin capture and DNA modification analysis. Several groups reported on the lack of specificity of some commercial, but unnamed, antibodies used for epigenetic studies.