Technetium-99m and rhenium-188 complexes with one and two pendant bisphosphonate groups for imaging arterial calcification

The synthesis of the first technetium-99m and rhenium-188 complexes with two pendant bisphosphonate groups is described, along with their in vivo use in imaging the skeleton and arterial calcification.</p

Imaging of changes in copper trafficking and redistribution in a mouse model of Niemann-Pick C disease using positron emission tomography

Niemann-Pick C disease (NPC) is an autosomal recessive lysosomal storage disorder resulting from mutations in the NPC1 (95% of cases) or NPC2 genes. Disturbance of copper homeostasis has been reported in NPC1 disease. In this study we have used whole-body positron emission tomography (PET) and brain electronic autoradiography with copper-64 (64Cu), in the form of the copper(II) bis(thiosemicarbazonato) complex 64Cu-GTSM, to image short-term changes in copper trafficking after intravenous injection in a transgenic mouse model of NPC1 disease. 64Cu-GTSM is taken up in all tissues and dissociates rapidly inside cells, allowing monitoring of the subsequent efflux and redistribution of 64Cu from all tissues. Significantly enhanced retention of 64Cu radioactivity was observed in brain, lungs and blood at 15 h post-injection in symptomatic Npc1−/− transgenic mice compared to wildtype controls. The enhanced retention of 64Cu in brain was confirmed by electronic autoradiography, particularly in the midbrain, thalamus, medulla and pons regions. Positron emission tomography imaging with 64Cu in selected chemical forms could be a useful diagnostic and research tool for the management and understanding of NPC1 disease

Protocols for Dual Tracer PET/SPECT Preclinical Imaging

BACKGROUND: Multi-tracer PET/SPECT imaging enables different modality tracers to be present simultaneously, allowing multiple physiological processes to be imaged in the same subject, within a short time-frame. Fluorine-18 and technetium-99m, two commonly used PET and SPECT radionuclides, respectively, possess different emission profiles, offering the potential for imaging one in the presence of the other. However, the impact of the presence of each radionuclide on scanning the other could be significant and lead to confounding results. Here we use combinations of (18)F and (99m)Tc to explore the challenges posed by dual tracer PET/SPECT imaging, and investigate potential practical ways to overcome them. METHODS: Mixed-radionuclide (18)F/(99m)Tc phantom PET and SPECT imaging experiments were carried out to determine the crossover effects of each radionuclide on the scans using Mediso nanoScan PET/CT and SPECT/CT small animal scanners. RESULTS: PET scan image quality and quantification were adversely affected by (99m)Tc activities higher than 100 MBq due to a high singles rate increasing dead-time of the detectors. Below 100 MBq (99m)Tc, PET scanner quantification accuracy was preserved. SPECT scan image quality and quantification were adversely affected by the presence of (18)F due to Compton scattering of 511 keV photons leading to over-estimation of (99m)Tc activity and increased noise. However, (99m)Tc:(18)F activity ratios of > 70:1 were found to mitigate this effect completely on the SPECT. A method for correcting for Compton scatter was also explored. CONCLUSION: Suitable combinations of injection sequence and imaging sequence can be devised to meet specific experimental multi-tracer imaging needs, with only minor or insignificant effects of each radionuclide on the scan of the other

Simple, mild, one-step labelling of proteins with gallium-68 using a tris(hydroxypyridinone) bifunctional chelator: a 68Ga-THP-scFv targeting the prostate-specific membrane antigen

Abstract Background Labelling proteins with gallium-68 using bifunctional chelators is often problematic because of unsuitably harsh labelling conditions such as low pH or high temperature and may entail post-labelling purification. To determine whether tris(hydroxypyridinone) (THP) bifunctional chelators offer a potential solution to this problem, we have evaluated the labelling and biodistribution of a THP conjugate with a new single-chain antibody against the prostate-specific membrane antigen (PSMA), an attractive target for staging prostate cancer (PCa). A single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, was prepared in order to achieve biokinetics matched to the half-life of gallium-68. The scFv, J591c-scFv, was engineered with a C-terminal cysteine. Results J591c-scFv was produced in HEK293T cells and purified by size-exclusion chromatography. A maleimide THP derivative (THP-mal) was coupled site-specifically to the C-terminal cysteine residue. The THP-mal-J591c-scFv conjugate was labelled with ammonium acetate-buffered gallium-68 from a 68Ge/68Ga generator at room temperature and neutral pH. The labelled conjugate was evaluated in the PCa cell line DU145 and its PSMA-overexpressing variant in vitro and xenografted in SCID mice. J591c-scFv was produced in yields of 4–6 mg/l culture supernatant and efficiently coupled with the THP-mal bifunctional chelator. Labelling yields > 95% were achieved at room temperature following incubation of 5 μg conjugate with gallium-68 for 5 min without post-labelling purification. 68Ga-THP-mal-J591c-scFv was stable in serum and showed selective binding to the DU145-PSMA cell line, allowing an IC50 value of 31.5 nM to be determined for unmodified J591c-scFv. Serial PET/CT imaging showed rapid, specific tumour uptake and clearance via renal elimination. Accumulation in DU145-PSMA xenografts at 90 min post-injection was 5.4 ± 0.5%ID/g compared with 0.5 ± 0.2%ID/g in DU145 tumours (n = 4). Conclusions The bifunctional chelator THP-mal enabled simple, rapid, quantitative, one-step room temperature radiolabelling of a protein with gallium-68 at neutral pH without a need for post-labelling purification. The resultant gallium-68 complex shows high affinity for PSMA and favourable in vivo targeting properties in a xenograft model of PCa

Development of [¹⁸F]FAMTO: A novel fluorine-18 labelled positron emission tomography (PET) radiotracer for imaging CYP11B1 and CYP11B2 enzymes in adrenal glands

Introduction: Primary aldosteronism accounts for 6\u201315% of hypertension cases, the single biggest contributor to global morbidity and mortality. Whilst ~50% of these patients have unilateral aldosterone-producing adenomas, only a minority of these have curative surgery as the current diagnosis of unilateral disease is poor. Carbon-11 radiolabelled metomidate ([11C]MTO) is a positron emission tomography (PET) radiotracer able to selectively identify CYP11B1/2 expressing adrenocortical lesions of the adrenal gland. However, the use of [11C]MTO is limited to PET centres equipped with on-site cyclotrons due to its short half-life of 20.4 min. Radiolabelling a fluorometomidate derivative with fluorine-18 (radioactive half life 109.8 min) in the para-aromatic position ([18F]FAMTO) has the potential to overcome this disadvantage and allow it to be transported to non-cyclotron-based imaging centres. Methods: Two strategies for the one-step radio-synthesis of [18F]FAMTO were developed. [18F]FAMTO was obtained via radiofluorination via use of sulfonium salt (1) and boronic ester (2) precursors. [18F]FAMTO was evaluated in vitro by autoradiography of pig adrenal tissues and in vivo by determining its biodistribution in rodents. Rat plasma and urine were analysed to determine [18F]FAMTO metabolites. Results: [18F]FAMTO is obtained from sulfonium salt (1) and boronic ester (2) precursors in 7% and 32% non-isolated radiochemical yield (RCY), respectively. Formulated [18F]FAMTO was obtained with >99% radiochemical and enantiomeric purity with a synthesis time of 140 min from the trapping of [18F]fluoride ion on an anion-exchange resin (QMA cartridge). In vitro autoradiography of [18F]FAMTO demonstrated exquisite specific binding in CYP11B-rich pig adrenal glands. In vivo [18F]FAMTO rapidly accumulates in adrenal glands. Liver uptake was about 34% of that in the adrenals and all other organs were <12% of the adrenal uptake at 60 min post-injection. Metabolite analysis showed 13% unchanged [18F]FAMTO in blood at 10 min post-administration and rapid urinary excretion. In vitro assays in human blood showed a free fraction of 37.5%. Conclusions: [18F]FAMTO, a new 18F-labelled analogue of metomidate, was successfully synthesised. In vitro and in vivo characterization demonstrated high selectivity towards aldosterone-producing enzymes (CYP11B1 and CYP11B2), supporting the potential of this radiotracer for human investigation

Effects of tillage and biomass on soil quality and productivity of lowland rice cultivation by small scale farmers in North Eastern India

•4-consecutive years results indicated that grain yield obtained with 2 spading+1 trampling+1 weeding (termed as minimum tillage-MT) was statistically at par with conventional tillage CT.•The weed biomass production was higher with MT than that under CT systems.•The amount of N, P and K recycled through rice straw was higher under CT, whereas, through weed biomass, it was higher under MT.•SOC, SMBC and dehydrogenase activity in MT were 5.7–13.3, 2.8–14.9 and 34.6–106.7%, respectively higher than CT.•The cost of cultivation increased with each increment in tillage. The net returns were higher with MT options. Intensive tillage-based production systems along with residue removal, grazing and/or burning of crop residues/biomass and poor nutrient replenishment through inadequate fertilizer and manure use are the major causes of soil degradation and unsustainable agriculture in hills of North Eastern India. Thus, a 4-year study (2003–07) was conducted during rainy (wet) seasons at Indian Council of Agricultural Research (ICAR) Research Complex for North Eastern Hill (NEH) Region, Umiam, India (950m a.s.l.). Objective of the study was to assess the effect of different tillage systems (individual or combinations of spading, trampling and hand weeding) on rice (Oryza sativa L.) productivity and soil (Typic Paleudalf) quality under in-situ residue management in lowland conditions. Transplanting in manually weeded unpuddled field was termed no-till (NT). In comparison, individual or combinations of spading, trampling (one partial manual puddling to incorporate weeds) and weeding was termed minimum tillage (MT). Treatment involving the maximum tillage included 4 spading+2 trampling+2 weedings, and was termed the conventional tillage (CT). The latter is practiced widely by farmers' in the region. Nine tillage treatments were laid out in a Randomized Block Design (RBD) and replicated thrice in a fixed plot size of 5m×5m. Increasing tillage intensity (combinations of spading along with trampling and weeding) increased grain yields. Agronomic yields obtained with 2 spading+1 trampling+1 weeding (MT option) was statistically (p=0.05) similar to that under CT. In general, the weed biomass was more under NT and MT than CT, and also contributed 2–4, 0.8–2 and 0.78–1.9 times more N, P and K, respectively, towards nutrient recycling than that under CT. However, the amount of N, P and K recycled through rice straw was more under CT than MT and NT. Weed biomass played a major role as a nutrient source in MT systems and contributed towards yield stabilization and improvement in soil quality. The soil organic carbon (25.2g/kg), soil microbial biomass carbon (198.7μg/g dry soil) and dehydrogenase activity (25.84μg TPF/g dry soil) in NT were 11.5, 17 and 107%, respectively, more than those under CT. These parameters under MT systems were 6–13, 2–15 and 35–88%, respectively, more compared to those under CT. The bulk density (ρb) under CT (1.18Mg/m3) was significantly higher than those observed under MT systems of only one spading or one trampling (1.15Mg/m3). The net return was the highest with MT system comprising of 2 spading+1 trampling+2 weeding ($367.5/ha), and that was 25.5% higher than that with CT. The net return per dollar (NRP) invested decreased with increasing tillage intensity. In-situ rice residue retention along with weed biomass recycling (MT) for 4-years improved soil quality, reduced cost of production and stabilized productivity in a low-input marginal (marginal soil and small scale farmers) hill agriculture

Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78 (Glucose-Regulated Protein, 78 kDa)-Loaded Extracellular Vesicles

OBJECTIVE: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. CONCLUSIONS: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs

Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78 (Glucose-Regulated Protein, 78 kDa)-Loaded Extracellular Vesicles

OBJECTIVE: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. CONCLUSIONS: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs

Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78 (Glucose-Regulated Protein, 78 kDa)-Loaded Extracellular Vesicles

OBJECTIVE: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. CONCLUSIONS: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs