Sirenomelia in Twin Pregnancy: A Case Report.

Sirenomelia is a rare congenital disorder that was once thought to be a severe case of caudal regression but is now thought to be entirely separate. It is often referred to as the "mermaid syndrome" because it causes the lower limbs to atrophy to varying degrees, giving the impression of a mermaid's tail or fin. The syndrome is often viewed as fatal due to the accompanying visceral deformities. Our case was a live born, delivered at term by caesarean section, to a 30-year-old third gravida having twin pregnancy. Examination of the baby revealed caudal dysgenesis with fusion of lower limbs, non-identifiable external genitalia and anus. The infant survived for 11 hours after birth. We report this case due to their rarity and term live birth. While sirenomelia is uncommon, the absence of distinct lower limbs on ultrasonography in the presence of oligo or anhydramnios may prompt consideration of the diagnosis of sirenomelia.

Immune Thrombocytopenia following COVID-19 Vaccine.

Several vaccines have been developed and are being administered against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Common side effects include fever, chills, headache, myalgia, and soreness at the injection site. However, some rare adverse effects have also been reported. We present a case of induced thrombocytopenia presenting with petechiae and mucosal bleeding which developed as an adverse response after first-dose administration of the Moderna COVID-19 vaccine.

68Ga-NODAGA-Exendin-4 PET/CT Improves the Detection of Focal Congenital Hyperinsulinism.

Surgery with curative intent can be offered to congenital hyperinsulinism (CHI) patients, provided that the lesion is focal. Radiolabeled exendin-4 specifically binds the glucagonlike peptide 1 receptor on pancreatic ß-cells. In this study, we compared the performance of 18F-DOPA PET/CT, the current standard imaging method for CHI, and PET/CT with the new tracer 68Ga-NODAGA-exendin-4 in the preoperative detection of focal CHI. Methods: Nineteen CHI patients underwent both 18F-DOPA PET/CT and 68Ga-NODAGA-exendin-4 PET/CT before surgery. The images were evaluated in 3 settings: a standard clinical reading, a masked expert reading, and a joint reading. The target (lesion)-to-nontarget (normal pancreas) ratio was determined using SUVmax Image quality was rated by pediatric surgeons in a questionnaire. Results: Fourteen of 19 patients having focal lesions underwent surgery. On the basis of clinical readings, the sensitivity of 68Ga-NODAGA-exendin-4 PET/CT (100%; 95% CI, 77%-100%) was higher than that of 18F-DOPA PET/CT (71%; 95% CI, 42%-92%). Interobserver agreement between readings was higher for 68Ga-NODAGA-exendin-4 than for 18F-DOPA PET/CT (Fleiss κ = 0.91 vs. 0.56). 68Ga-NODAGA-exendin-4 PET/CT provided significantly (P = 0.021) higher target-to-nontarget ratios (2.02 ± 0.65) than did 18F-DOPA PET/CT (1.40 ± 0.40). On a 5-point scale, pediatric surgeons rated 68Ga-NODAGA-exendin-4 PET/CT as superior to 18F-DOPA PET/CT. Conclusion: For the detection of focal CHI, 68Ga-NODAGA-exendin-4 PET/CT has higher clinical sensitivity and better interobserver correlation than 18F-DOPA PET/CT. Better contrast and image quality make 68Ga-NODAGA-exendin-4 PET/CT superior to 18F-DOPA PET/CT in surgeons' intraoperative quest for lesion localization.

The Development of Cognate Awareness in Child Second/Third Language Learners of French in French Immersion: The Effects of Orthographic Overlap and Cognate Status.

Purpose Our 1-year longitudinal study tracked the development of cognate awareness among second (L2) and third language (L3) learners of French in French immersion in Grades 1 and 2 to explore the impact of orthographic overlap and cognate status (true vs. false) on children's ability to recognize cognate relationships. We also assessed the impact of French L2/L3 status on performance. Method We compared performance on three conditions (true cognates with same and similar spellings, false cognates with same spellings) within and across grades. We used a direct measure of cognate awareness that required children (n = 81) to distinguish true from false cognates presented orally and in print. Results Overall, Grade 1 children failed to recognize cognate relationships between true cognates with similar spellings, but successfully recognized true cognates with same spellings. Performance on all conditions increased significantly between Grades 1 and 2. The greatest improvement was seen on true cognates with similar spellings. Performance on false cognates was inferior to performance on true cognates with same spellings in Grade 1, and inferior to performance on both same and similar spelled true cognates in Grade 2. No differences were found due to L2/L3 status. Conclusions Among sequential learners of L2/L3 French in the early stages of additional language learning, cognate awareness is impacted by the degree of orthographic overlap, as well as by cognate status. Children's ability to recognize cross-language orthographic and semantic relationships improves substantially across the early elementary grades. Supplemental Material https://doi.org/10.23641/asha.16821106.

Long-circulating XTEN864-annexin A5 fusion protein for phosphatidylserine-related therapeutic applications.

Annexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of 111In-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE-/- mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug.

SPECT/CT Imaging, Biodistribution and Radiation Dosimetry of a 177Lu-DOTA-Integrin αvß6 Cystine Knot Peptide in a Pancreatic Cancer Xenograft Model.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin αvß6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin αvß6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin αvß6-specific knottin molecular probe containing the therapeutic radionuclide 177Lu for targeting of PDAC. METHODS: The expression of integrin αvß6 in PDAC cell lines BxPC-3 and Capan-2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC-3 and Capan-2 cell lines as well as the maximum number of binding sites in these cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan-2 xenograft tumor mouse model. RESULTS: RT-qPCR and immunofluorescence results showed high expression of integrin αvß6 in BxPC-3 and Capan-2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC-3 and Capan-2, respectively. SPECT/CT and biodistribution analysis of the conjugate 177Lu-DOTA-integrin αvß6 knottin demonstrated accumulation in Capan-2 xenograft tumors (3.13 ± 0.63%IA/g at day 1 post injection) with kidney uptake at 19.2 ± 2.5 %IA/g, declining much more rapidly than in tumors. CONCLUSION: 177Lu-DOTA-integrin αvß6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy.

In Comparison to PSA, Interim Ga-68-PSMA PET/CT Response Evaluation Based on Modified RECIST 1.1 After 2nd Cycle Is Better Predictor of Overall Survival of Prostate Cancer Patients Treated With 177Lu-PSMA.

BACKGROUND: Prostate-specific membrane antigen (PSMA) targeting radioligands have transformed treatment of prostate cancer. Radioligand therapy (RLT) with 177Lu-PSMA in metastasized castration resistant prostate cancer (mCRPC) achieves objective response and disease stabilization in roughly two third of patients, whereas one third of patients progress. This study was performed to assess the role of interim PSMA PET/CT after the 2nd cycle of RLT for early prediction of overall survival in patients undergoing RLT with 177Lu-PSMA. METHODS: 38 mCRPC patients (68.9 ± 8.1 y) treated with at least two cycles of RLT at 8 week intervals and interim 68Ga-PSMA PET/CT (PET) at 8-10 weeks after the 2nd cycle of RLT were included in this study. Prostate-specific antigen (PSA) response was evaluated according to the Prostate Cancer Working Group 3 criteria. Radiographic response assessment of soft tissue, lymph node, and bone lesions was performed according to RECIST 1.1 including the PET component. Patients' data were collected for follow-up from the local Comprehensive Cancer Center Register. RESULTS: Median follow-up was 19.7 months (4.7-45.3). PSA response after the 2nd therapy cycle showed partial remission (PR) in 23.7%, stable disease (SD) in 50%, and progressive disease (PD) in 26.3% of patients. In comparison, 52.6, 23.7, and 23.7% of patients showed PR, SD, and PD respectively on PET/CT. The strength of agreement between PSA response and PET/CT response criteria was only fair (kappa 0.346). Median overall survival (OS) was 22.5 months (95% CI: 15.8-29.2). Median OS stratified to PSA/PET response was 25.6/25.6 months for PR, 21.7/30.6 months for SD and 19.4/13.1 months for PD (p = 0.496 for PSA and 0.013 for PET/CT response). CONCLUSIONS: Interim PSMA PET/CT based response evaluation at 8-10 weeks after the 2nd cycle of RLT is predictive of overall survival and PD in patients treated with 177Lu-PSMA. On the contrary, PSA appears to have only limited predictive value.

Effect of Peptide Dose on Radiation Dosimetry for Peptide Receptor Radionuclide Therapy with 177Lu-DOTATOC: A Pilot Study.

BACKGROUND: Optimal peptide concentration in treatment with 177Lu-DOTATOC/DOTATATE is a matter of debate. Most of the studies with peptide receptor radionuclide therapy mention peptide dose ranging between 100 and 250 µg. The aim of this is to identify possible differences in radiation-absorbed doses (D/Gy) to tumor and kidney as a function of the peptide mass dose in order to identify the most suitable peptide dose for treatment. The therapeutic index (Dtumor/Dkidneys) was assessed as a key parameter for the treatment response. MATERIALS AND METHODS: Five patients with metastasized Grade 1 to Grade 2 neuroendocrine tumor were analyzed in this study. Patients (n = 4) received two cycles of treatment with intravenously injected 177Lu-DOTATOC containing peptide mass doses of 200 µg and 90 µg, alternatively; one patient was treated with 90 µg peptide mass in both the therapy cycles. Whole-body (head to mid-thigh) three-dimensional single-photon emission computerized tomography (3D SPECT)/CT images were acquired at 1, 4, 24, 48, and 72 h following the injection of 177Lu-DOTATOC. Attenuation correction for 3D SPECT images was performed using CT data acquired and fused with the SPECT data (SPECT/CT). RESULTS: Overall, 28 target lesions (liver n = 17, lung n = 4, lymph nodes n = 1, and bone n = 2) were analyzed after 1st and 2nd therapy cycles. Tumor normalized absorbed doses varied by a factor of 74 between 0.35 and 26 mGy/MBq. Averaged over all patients, a higher normalized mean tumor dose (10.51 mGy/MBq) was achieved for a peptide dose of 200 µg compared to 90 µg (4.58 mGy/MBq). Kidneys doses varied by a factor of up to 4 between patients (0.25-1.0 mGy/MBq) (independent of dose cycle and peptide dose) and by a factor of up to 2 between dose cycles. The mean kidney dose was 13.7% higher for the 90 µg peptide dose compared to 200 µg. Given the higher tumor dose, the mean therapeutic index of a 200 µg mass dose was considerably higher (16.95), compared to a 90 µg mass dose (9.63). This coincided with the observation, that lesion volume reduction was more pronounced after an initial treatment with a 200 µg mass dose. Biologically effective dose was only 5. 1%-19.3% higher than the absorbed dose for individual dose cycles. CONCLUSIONS: Higher peptide dose of 200 µg appears to be more suitable than 90 µg in terms of tumor dose, kidney dose, and therapeutic index for treatment with 177Lu-DOTATOC.

Multimodal Imaging of 2-Cycle PRRT with 177Lu-DOTA-JR11 and 177Lu-DOTATOC in an Orthotopic Neuroendocrine Xenograft Tumor Mouse Model.

Peptide receptor radionuclide therapy (PRRT) using radiolabeled somatostatin receptor (SSTR) analogs is a common approach in advanced neuroendocrine neoplasms. Recently, SSTR antagonists have shown promising results for imaging and therapy due to a higher number of binding sites than in commonly used agonists. We evaluated PRRT with SSTR agonist 177Lu-DOTATOC and antagonist 177Lu-DOTA-JR11 longitudinally in an orthotopic murine pancreatic neuroendocrine neoplasm model expressing human SSTR2. Morphologic and metabolic changes during treatment were assessed using multimodal imaging, including hybrid PET/MRI and SPECT/CT. Methods: In vitro radioligand binding and internalization assays and cell-cycle analysis were performed. SSTR2-transfected BON cells (BON-SSTR2) were used for in vivo experiments. Tumor-bearing mice received 2 intravenous injections of 100 µL of saline, 30 MBq of 177Lu-DOTATOC, or 20 MBq of 177Lu-DOTA-JR11 with an interval of 3 wk. Weekly T2-weighted MRI was performed for tumor monitoring. Viability of the tumor tissue was assessed by 18F-FDG PET/MRI once after PRRT. Tumor and kidney uptake of the respective radiopharmaceuticals was measured 24 h after injection by SPECT/CT. Results: Compared with 177Lu-DOTATOC, 177Lu-DOTA-JR11 treatment resulted in an increased accumulation of cells in G2/M phase. Animals treated with the SSTR antagonist showed a significant reduction in tumor size (P < 0.001) and an increased median survival (207 d; interquartile range [IQR], 132-228) compared with 177Lu-DOTATOC (126 d; IQR, 118-129). SPECT/CT revealed a 4-fold higher median tumor uptake for the antagonist and a 3-fold higher tumor-to-kidney ratio in the first treatment cycle. During the second therapy cycle, tumor uptake of 177Lu-DOTATOC was significantly lower (P = 0.01) whereas 177Lu-DOTA-JR11 uptake remained stable. Imaging of tumor morphology indicated comparatively larger necrotic fractions for 177Lu-DOTA-JR11 despite further tumor growth. These results were confirmed by 18F-FDG PET, revealing the least amount of viable tumor tissue in 177Lu-DOTA-JR11-treated animals, at 6.2% (IQR, 2%-23%). Conclusion:177Lu-DOTA-JR11 showed a higher tumor-to-kidney ratio and a more pronounced cytotoxic effect than did 177Lu-DOTATOC. Additionally, tumor uptake was more stable over the course of 2 treatment cycles.

A Cyanine-Bridged Somatostatin Hybrid Probe for Multimodal SSTR2 Imaging in Vitro and in Vivo: Synthesis and Evaluation.

Multimodal imaging probes have attracted the interest of ongoing research, for example, for the surgical removal of tumors. Modular synthesis approaches allow the construction of hybrid probes consisting of a radiotracer, a fluorophore and a targeting unit. We present the synthesis of a new asymmetric bifunctional cyanine dye that can be used as a structural and functional linker for the construction of such hybrid probes. 68 Ga-DOTATATE, a well-characterized radiopeptide targeting the overexpressed somatostatin receptor subtype 2 (SSTR2) in neuroendocrine tumors, was labeled with our cyanine dye, thus providing additional information along with the data obtained from the radiotracer. We tested the SSTR2-targeting and imaging properties of the resulting probe 68 Ga-DOTA-ICC-TATE in vitro and in a tumor xenograft mouse model. Despite the close proximity between dye and pharmacophore, we observed a high binding affinity towards SSTR2 as well as elevated uptake in SSTR2-overexpressing tumors in the positron emission tomography (PET) scan and histological examination.

Radixin modulates the function of outer hair cell stereocilia.

The stereocilia of the inner ear sensory cells contain the actin-binding protein radixin, encoded by RDX. Radixin is important for hearing but remains functionally obscure. To determine how radixin influences hearing sensitivity, we used a custom rapid imaging technique to visualize stereocilia motion while measuring electrical potential amplitudes during acoustic stimulation. Radixin inhibition decreased sound-evoked electrical potentials. Other functional measures, including electrically induced sensory cell motility and sound-evoked stereocilia deflections, showed a minor amplitude increase. These unique functional alterations demonstrate radixin as necessary for conversion of sound into electrical signals at acoustic rates. We identified patients with RDX variants with normal hearing at birth who showed rapidly deteriorating hearing during the first months of life. This may be overlooked by newborn hearing screening and explained by multiple disturbances in postnatal sensory cells. We conclude radixin is necessary for ensuring normal conversion of sound to electrical signals in the inner ear.

Relationship of Renal Function in Mice to Strain, Sex and 177Lutetium-Somatostatin Receptor Ligand Treatment.

AIM: Aim of the study was to establish parameters for 99mTc-MAG3 SPECT renal uptake kinetics in healthy SCID mice as a function of mouse strain and sex and to evaluate the feasibility of this method for detecting 177Lu-somatostatin receptor ligand (177Lu-SRL) treatment effects on kidney function. MATERIALS AND METHODS: Dynamic semi-stationary SPECT acquisitions (68 frames, total duration 35 min) was started prior to i. v. injection of 99mTc-MAG3 in 12 female and 12 male SCID mice. Additionally, 6 female SCID mice with neuroendocrine tumors were imaged 1-5 months after 177Lu-SRL (5 DOTATOC, 1 DOTA-JR11) treatment. Kidney function is expressed as maximum time to peak (Tmax), T50 and T25 in minutes (median [interquartile range]). Differences between groups were tested using the Mann-Whitney-U test, and SCID mouse parameters were compared with data for C57BL/6N mice from a recent publication. RESULTS: Significant sex-based differences in Tmax between strains were observed (females: C57BL/6N 1.6 [1.4-1.7], SCID 1.4 [1.3-1.5], p = 0.05; males: C57BL/6N 1.4 [1.3-1.4], SCID 1.6 [1.4-1.7], p = 0.04). In C57BL/6N mice, females showed a later Tmax (p < 0.01) than males. SCID mice showed no difference (p = 0.14). Treated SCID mice showed no significant delay in Tmax (2.0 [1.4-2.7], p = 0.15) but a significant delay in T50 (p = 0.02) and T25 (p = 0.01) compared to healthy untreated mice. CONCLUSION: This study demonstrated significant sex-related differences between SCID and C57BL/6N mouse strains in kidney function. Establishment of normal values for different strains and sexes therefore is important for experimental therapy studies. Renal SPECT imaging with 99mTc-MAG3 was sufficiently sensitive to detect 177Lu-SRL treatment toxic effects on kidney function in SCID mice.

CMKLR1-targeting peptide tracers for PET/MR imaging of breast cancer.

Background: Molecular targeting remains to be a promising approach in oncology. Overexpression of G protein-coupled receptors (GPCRs) in human cancer is offering a powerful opportunity for tumor-selective imaging and treatment employing nuclear medicine. We utilized novel chemerin-based peptide conjugates for chemokine-like receptor 1 (CMKLR1) targeting in a breast cancer xenograft model. Methods: By conjugation with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of five highly specific, high-affinity tracers for hybrid positron emission tomography/magnetic resonance (PET/MR) imaging. A xenograft model with target-positive DU4475 and negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging in vivo. We acquired small animal PET/MR images, assessed biodistribution by ex vivo measurements and investigated the tracer specificity by blocking experiments. Results: Five CMKLR1-targeting peptide tracers demonstrated high biological activity and affinity in vitro with EC50 and IC50 values below 2 nM. Our target-positive (DU4475) and target-negative (A549) xenograft model could be validated by ex vivo analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three most promising tracers [68Ga]Ga-DOTA-AHX-CG34, [68Ga]Ga-DOTA-KCap-CG34 and [68Ga]Ga-DOTA-ADX-CG34 with best tumor uptake were further analyzed. Hybrid PET/MR imaging along with concomitant biodistribution studies revealed distinct CMKLR1-specific uptake (5.1% IA/g, 3.3% IA/g and 6.2% IA/g 1 h post-injection) of our targeted tracers in DU4475 tumor tissue. In addition, tumor uptake was blocked by excess of unlabeled peptide (6.4-fold, 5.5-fold and 3.4-fold 1 h post-injection), further confirming CMKLR1 specificity. Out of five tracers, we identified these three tracers with moderate, balanced hydrophilicity to be the most potent in receptor-mediated tumor targeting. Conclusion: We demonstrated the applicability of 68Ga-labeled peptide tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR imaging, paving the way for developing them into theranostics for tumor treatment.

Attenuated palmitoylation of serotonin receptor 5-HT1A affects receptor function and contributes to depression-like behaviors.

The serotonergic system and in particular serotonin 1A receptor (5-HT1AR) are implicated in major depressive disorder (MDD). Here we demonstrated that 5-HT1AR is palmitoylated in human and rodent brains, and identified ZDHHC21 as a major palmitoyl acyltransferase, whose depletion reduced palmitoylation and consequently signaling functions of 5-HT1AR. Two rodent models for depression-like behavior show reduced brain ZDHHC21 expression and attenuated 5-HT1AR palmitoylation. Moreover, selective knock-down of ZDHHC21 in the murine forebrain induced depression-like behavior. We also identified the microRNA miR-30e as a negative regulator of Zdhhc21 expression. Through analysis of the post-mortem brain samples in individuals with MDD that died by suicide we find that miR-30e expression is increased, while ZDHHC21 expression, as well as palmitoylation of 5-HT1AR, are reduced within the prefrontal cortex. Our study suggests that downregulation of 5-HT1AR palmitoylation is a mechanism involved in depression, making the restoration of 5-HT1AR palmitoylation a promising clinical strategy for the treatment of MDD.

Serotonin receptor oligomerization regulates cAMP-based signaling.

Protein-protein interaction is often investigated using quantitative molecular microscopy with Förster resonant energy transfer (FRET). Here, we combined 'linear unmixing FRET' (lux-FRET) with the simultaneous application of a FRET-based biosensor for cAMP to investigate the oligomerization between the 5-HT7 receptor (5-HT7R, also known as HTR7) and the 5-HT1A receptor (5-HT1AR, also known as HTR1A) and its importance for cAMP signaling. We found that the 5-HT7R not only stimulates cAMP production, but also forms hetero-oligomers with 5-HT1AR, which blocks the inhibitory effect of the latter. 5-HT7R signaling, however, is not affected by this hetero-oligomerization. By modeling the kinetics of intracellular cAMP level changes in relation to the 5-HT7R:5-HT1AR stoichiometry, we were able to decipher the complex signaling characteristics of endogenous serotonin receptors in cultured hippocampal neurons. Our findings indicate that serotonergic signaling is not only modulated by the concentration of an individual receptor but also by its specific interaction with other receptors in endogenous systems. We conclude that the regulated ratio of serotonin receptors in immature and mature neurons may be critically involved in both the onset and response to treatments of psychiatric diseases, such as anxiety and depression.

Increasing molar activity by HPLC purification improves 68Ga-DOTA-NAPamide tumor accumulation in a B16/F1 melanoma xenograft model.

PURPOSE: Melanocortin receptor 1 (MC1R) is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium (68Ga) labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography (PET) imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide. PROCEDURES: The MC1R ligand DOTA-NAPamide was labeled with 68Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68Ga (95°C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors. RESULTS: In biodistribution studies, non-purified 68Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracer preparations. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. CONCLUSIONS: We demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.

Control of hearing sensitivity by tectorial membrane calcium.

When sound stimulates the stereocilia on the sensory cells in the hearing organ, Ca2+ ions flow through mechanically gated ion channels. This Ca2+ influx is thought to be important for ensuring that the mechanically gated channels operate within their most sensitive response region, setting the fraction of channels open at rest, and possibly for the continued maintenance of stereocilia. Since the extracellular Ca2+ concentration will affect the amount of Ca2+ entering during stimulation, it is important to determine the level of the ion close to the sensory cells. Using fluorescence imaging and fluorescence correlation spectroscopy, we measured the Ca2+ concentration near guinea pig stereocilia in situ. Surprisingly, we found that an acellular accessory structure close to the stereocilia, the tectorial membrane, had much higher Ca2+ than the surrounding fluid. Loud sounds depleted Ca2+ from the tectorial membrane, and Ca2+ manipulations had large effects on hair cell function. Hence, the tectorial membrane contributes to control of hearing sensitivity by influencing the ionic environment around the stereocilia.

Analysis of receptor-receptor interaction by combined application of FRET and microscopy.

G protein-coupled receptors (GPCRs) participate in the regulation of many cellular processes and, therefore, represent key targets for pharmacological treatment. The existence of GPCR homo- and heterodimers has become generally accepted, and a growing body of evidence points to the functional importance of oligomeric complexes for the receptor trafficking, receptor activation, and G protein coupling in native tissues. Quantitative molecular microscopy is becoming more and more important to investigate such receptor-receptor interaction in their native environments. Förster resonance energy transfer (FRET) is thereby utilized to aim at investigating the interaction of molecules at distances beyond diffraction-limited spatial resolution. The exact determination of the FRET signals, which are often only fractions of the fluorescence signals, requires extensive experimental effort. Moreover, the correct interpretation of FRET measurements as well as FRET data-based modeling represents an essential challenge in microscopy and biophysics. In this chapter, we present and discuss variety of acquisition protocols and models based on "linear unmixing FRET" (lux-FRET) to investigate receptor-receptor interaction in living cells with high spatial and temporal resolution. Here, we show how to apply lux-FRET in spectroscopic and different imaging devices, based either on spectral detection or on filter cubes. We focus on detailed description for FRET measurements and analyses based on sophisticated acquisition procedures according to different experimental setups and also provide several examples of biological applications.

Peptide translocation through the mesoscopic channel: binding kinetics at the single molecule level.

Single channel electrophysiological studies have been carried out to elucidate the underlying interactions during the translocation of polypeptides through protein channels. For this we used OmpF from the outer cell membrane of E. coli and arginine-based peptides of different charges, lengths and covalently linked polyethylene glycol as a model system. In order to reveal the fast kinetics of peptide binding, we performed a temperature scan. Together with the voltage-dependent single-channel conductance, we quantify peptide binding and translocation.