Novel [68Ga/177Lu]Ga/Lu-AZ-093 as PSMA-Targeting Agent for Diagnosis and Radiotherapy.

Prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer cells can serve as a target for imaging and radioligand therapy (RLT). Previously, [68Ga]Ga-P16-093, containing a Ga(III) chelator, N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC), displayed excellent PSMA-targeting properties and showed a high tumor uptake and retention useful for diagnosis in prostate cancer patients. Recently, [177Lu]Lu-PSMA-617 has been approved by the U.S. food and drug administration (FDA) for the treatment of prostate cancer patients. Derivatives of PSMA-093 using AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), as the chelator, were designed as alternative agents forming complexes with both diagnostic and therapeutic radiometals, such as gallium-68 (log K = 22.18) or lutetium-177 (log K = 21.85). The aim of this study is to evaluate AAZTA-Gly-O-(methylcarboxy)-Tyr-Phe-Lys-NH-CO-NH-Glu (designated as AZ-093, 1) leading to a gallium-68/lutetium-177 theranostic pair as potential PSMA targeting agents. Synthesis of the desired precursor, AZ-093, 1, was effectively accomplished. Labeling with either [68Ga]GaCl3 or [177Lu]LuCl3 in a sodium acetate buffer solution (pH 4-5) at 50 °C in 5 to 15 min produced either [68Ga]Ga-1 or [177Lu]Lu-1 with high yields and excellent radiochemical purities. Results of in vitro binding studies, cell uptake, and retention (using PSMA-positive prostate carcinoma cells line, 22Rv1-FOLH1-oe) were comparable to that of [68Ga]Ga-P16-093 and [177Lu]Lu-PSMA-617, respectively. Specific cellular uptake was determined with or without the competitive blocking agent (2 µM of "cold" PSMA-11). Cellular binding and internalization showed a time-dependent increase over 2 h at 37 °C in the PSMA-positive cells. The cell uptakes were completely blocked by the "cold" PSMA-11 suggesting that they are competing for the same PSMA binding sites. In the mouse model with implanted PSMA-positive tumor cells, both [68Ga]Ga-1 and [177Lu]Lu-1 displayed excellent uptake and retention in the tumor. Results indicate that [68Ga]Ga/[177Lu]Lu-1 (68Ga]Ga/[177Lu]Lu-AZ-093) is potentially useful as PSMA-targeting agent for both diagnosis and radiotherapy of prostate cancer.

Comparison of novel PSMA-targeting [177Lu]Lu-P17-087 with its albumin binding derivative [177Lu]Lu-P17-088 in metastatic castration-resistant prostate cancer patients: a first-in-human study.

PURPOSE: Prostate-specific membrane antigen (PSMA) is a promising target for diagnosis and radioligand therapy (RLT) of prostate cancer. Two novel PSMA-targeting radionuclide therapy agents, [177Lu]Lu-P17-087, and its albumin binder modified derivative, [177Lu]Lu-P17-088, were evaluated in metastatic castration-resistant prostate cancer (mCRPC) patients. The primary endpoint was dosimetry evaluation, the second endpoint was radiation toxicity assessment (CTCAE 5.0) and PSA response (PCWG3). METHODS: Patients with PSMA-positive tumors were enrolled after [68Ga]Ga-PSMA-11 PET/CT scan. Five mCRPC patients received [177Lu]Lu-P17-087 and four other patients received [177Lu]Lu-P17-088 (1.2 GBq/patient). Multiple whole body planar scintigraphy was performed at 1.5, 4, 24, 48, 72, 120 and 168 h after injection and one SPECT/CT imaging was performed at 24 h post-injection for each patient. Dosimetry evaluation was compared in both patient groups. RESULTS: Patients showed no major clinical side-effects under this low dose treatment. As expected [177Lu]Lu-P17-088 with longer blood circulation (due to its albumin binding) exhibited higher effective doses than [177Lu]Lu-P17-087 (0.151 ± 0.036 vs. 0.056 ± 0.019 mGy/MBq, P = 0.001). Similarly, red marrow received 0.119 ± 0.068 and 0.048 ± 0.020 mGy/MBq, while kidney doses were 0.119 ± 0.068 and 0.046 ± 0.022 mGy/MBq, respectively. [177Lu]Lu-P17-087 demonstrated excellent tumor uptake and faster kinetics; while [177Lu]Lu-P17-088 displayed a slower washout and higher average dose (7.75 ± 4.18 vs. 4.72 ± 2.29 mGy/MBq, P = 0.018). After administration of [177Lu]Lu-P17-087 and [177Lu]Lu-P17-088, 3/5 and 3/4 patients showed reducing PSA values, respectively. CONCLUSION: [177Lu]Lu-P17-088 and [177Lu]Lu-P17-087 displayed different pharmacokinetics but excellent PSMA-targeting dose delivery in mCRPC patients. These two agents are promising RLT agents for personalized treatment of mCRPC. Further studies with increased dose and frequency of RLT are warranted to evaluate the potential therapeutic efficacy. TRIAL REGISTRATION: 177Lu-P17-087/177Lu-P17-088 in Patients with Metastatic Castration-resistant Prostate Cancer (NCT05603559, Registered at 25 October, 2022). URL OF REGISTRY: https://classic. CLINICALTRIALS: gov/ct2/show/NCT05603559 .

Serotonin transporter imaging agent as a probe for ß-cells of pancreas.

OBJECTIVE: Diabetes mellitus (DM) is one of the major diseases in the world. Nuclear medicine imaging may be able to detect functional status of pancreatic ß cells in vivo, which might elucidate the pathological mechanisms of diabetes and develop individualized treatment plans. In this study, we evaluated the ability of [125I]ADAM, a serotonin transporter (SERT) imaging agent, as a probe for detecting pancreatic ß-cell mass (BCM). METHODS: In vitro cell studies were evaluated in INS-1 cells (rat islet ß cell line). Biodistribution studies were performed in male normal Sprague-Dawley rats and alloxan-induced type 1 diabetes mellitus (T1DM) rats. Distribution and expression of SERT protein in pancreas of rats were also measured by immunofluorescence staining and Western blot. RESULTS: In vitro cell studies showed that the concentration of [125I]ADAM associated with the INS-1 cells was increased gradually with incubation time, and the SERT specific inhibitor, escitalopram, exhibited the inhibitory effect on this interaction. Biodistribution studies also showed that the uptake of [125I]ADAM in the pancreas of normal rats was decreased in the presence of escitalopram. However, in the T1DM rat model with a significant ß cells reduction, the uptake of pancreas was increased when compared with the control. Through immunofluorescence staining and Western blot, it was found that both the endocrine and exocrine cells of the normal pancreas expressed SERT protein, and the level of SERT protein in the exocrine cells was higher than islets. In the diabetic state, the expression of SERT in the exocrine cells was further increased. CONCLUSIONS: The SERT imaging agent, [125I]ADAM, at the present form will not be suitable for imaging ß cells, specifically because there were extraordinarily high non-specific signals contributing from the exocrine cells of pancreas. In addition, we noticed that the level of SERT expression was abnormally elevated in the diabetic state, which might provide an unexpected target for studying the pathological mechanisms of diabetes.

Free Energy Landscape of Type III Fibronectin Domain with Identified Intermediate State and Hierarchical Symmetry.

The tenth domain of type III fibronectin (FNIII_{10}) mediates cell adhesion to the extracellular matrix. Despite its structural similarity to immunoglobulin domains, FNIII_{10} exhibits unique unfolding behaviors. We employed magnetic tweezers to investigate the unfolding and folding dynamics of FNIII_{10} under physiological forces (4-50 pN). Our results showed that FNIII_{10} follows a consistent transition pathway with an intermediate state characterized by detached A and G ß strands. We determined the folding free energies and all force-dependent transition rates of FNIII_{10} and found that both unfolding rates from the native state to the intermediate state and from the intermediate state to the unfolded state deviate from Bell's model. We constructed a quantitative free energy landscape with well-defined traps and barriers that exhibits a hierarchical symmetrical pattern. Our findings provide a comprehensive understanding of FNIII_{10} conformational dynamics and demonstrate how free energy landscape of multistate biomolecules can be precisely mapped, illuminating the relationship between thermal stability, intermediate states, and folding rates in protein folding.

[68Ga]Ga-HBED-CC-FAPI Derivatives with Improved Radiolabeling and Specific Tumor Uptake.

Fibroblast activation protein (FAP) is selectively expressed in tumors and highly important for maintaining the microenvironment in malignant tumors. Radioisotope-labeled FAP inhibitors (FAPIs) were proven to be useful for diagnosis and radionuclide therapy of cancer and are under active clinical investigations. Ga-HBED complex displays a higher in vivo stability constant (log KGaL: 38.5), compared to that of Ga-DOTA (log KGaL: 21.3). Such advantage in stability constant suggests that it may be useful for development of alternative FAPI imaging agents. In this study, previously reported [68Ga]Ga-DOTA-FAPI-02 and -04 were converted to the corresponding [68Ga]Ga-HBED-CC-FAPI-02 and -04 derivatives ([68Ga]Ga-4, [68Ga]Ga-5, [68Ga]Ga-6, and [68Ga]Ga-7). It was found that substituting the DOTA chelating group with HBED-CC led to several unique and desirable tumor-targeting properties: (1) robust, fast, and high yield labeling─readily adaptable to a kit formulation; (2) high stabilities in vitro; (3) excellent FAP binding affinities (IC50 ranging between 4 and 7 nM) and improved cell uptake and retention (in HT1080 (FAP+) cells); and (4) excellent selective in vivo tumor uptake in nude mice bearing U87MG tumor. It appeared that Ga(III) chelation with HBED-CC improved the in vivo kinetics favoring higher tumor uptake and retention compared to the corresponding Ga-DOTA complex. Out of the four tested ligands the new [68Ga]Ga-HBED-CC-FAPI dimer, [68Ga]Ga-6, displayed the best tumor localization properties, and further studies are warranted to demonstrate that it is an alternative FAP imaging agent for cancer patients.

Head-to-Head Comparison of 68 Ga-P16-093 and 68 Ga-PSMA-617 PET/CT in Patients With Primary Prostate Cancer : A Pilot Study.

PURPOSE: We aimed to compare the diagnostic performance of 68 Ga-P16-093 and 68 Ga-PSMA-617 PET/CT in primary prostate cancer (PCa) patients. PATIENTS AND METHODS: Thirty untreated primary PCa patients were enrolled. Each patient underwent 68 Ga-P16-093 and 68 Ga-PSMA-617 PET/CT within a week. In addition to visual analysis, SUV was measured for semiquantitative comparison and correlation analysis. RESULTS: 68 Ga-P16-093 PET/CT detected more positive tumors than 68 Ga-PSMA-617 PET/CT (67 vs 56, P = 0.002), especially for intraprostatic lesions (29 vs 24, P = 0.025) and lymph node metastases (23 vs 17, P = 0.034). Further, 68 Ga-P16-093 PET/CT exhibited significantly higher SUV max of matched tumors (18.3 ± 14.4 vs 13.9 ± 11.8, P < 0.001). Besides, the SUV max of high-risk patients (based on D'Amico classification) on 68 Ga-P16-093 PET/CT was significantly higher than that of low- and intermediate-risk PCa patients (20.9 ± 9.9 vs 8.9 ± 9.1 vs 10.1 ± 5.2, P = 0.007). The SUV max of tumor measured by 68 Ga-P16-093 PET/CT had a moderate association with biopsy Gleason score ( r = 0.462, P = 0.005) and prostate-specific antigen value ( r = 0.491, P = 0.002), and significantly correlated with PSMA expression ( r = 0.732, P < 0.001). CONCLUSIONS: 68 Ga-P16-093 PET/CT exhibited higher tumor uptake and potentially better tumor detection capability than 68 Ga-PSMA-617 PET/CT, which suggested that 68 Ga-P16-093 may be more suitable in the diagnosis and staging of primary PCa patients.

In vivo and in vitro binding of [125 I]I-R-(+)-TISCH: A dopamine D1 receptor ligand for studying pancreatic ß-cell mass.

Diabetes mellitus (DM) and insulinoma are mainly affected by the status of pancreatic ß-cell mass (BCM). Development of imaging agents for BCM allows to study pancreatic ß cells and the relationship between ß cells and DM or insulinoma. In this study, we investigated the density of dopamine D1 receptor on the ß cells and measured BCM by statistical image processing. The pancreatic uptakes of [125 I]I-R-(+)-7-chloro-8-hydroxy-1-(3'-iodopheny1)-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([125 I]I-R-(+)-TISCH), dopamine D1 receptor tracer, in normal and diabetic rats displayed significant differences at 30 min (1.11 ± 0.08% ID/g vs. 0.63 ± 0.09% ID/g, p < 0.0001). In the presence of SCH23390, the pancreatic uptake of [125 I]I-R-(+)-TISCH at 30 min in normal rats was lower (1.01 ± 0.04% ID/g, p < 0.05). Although the blocking was not complete, [125 I]I-R-(+)-TISCH showed specific binding signals to the pancreas. Furthermore, the uptakes of [125 I]I-R-(+)-TISCH in INS-1 cells were reduced in the presence of SCH23390 at different concentrations. [125 I]I-R-(+)-TISCH displayed a respectable uptake in insulinoma. Overall, [125 I]I-R-(+)-TISCH provided specific binding signals to pancreatic ß cells. Although the specific signal may not be sufficient for imaging in vivo, the dopamine D1 receptor can still be considered as a potential target for studying BCM. Further investigation will be required to optimize the ligand.

Free-energy landscape of two-state protein acylphosphatase with large contact order revealed by force-dependent folding and unfolding dynamics.

Acylphosphatase (AcP) is a small protein with 98 amino acid residues that catalyzes the hydrolysis of carboxyl-phosphate bonds. AcP is a typical two-state protein with slow folding rate due to its relatively large contact order in the native structure. The mechanical properties and unfolding behavior of AcP has been studied by atomic force microscope. Here using stable magnetic tweezers, we measured the force-dependent folding rates within a force range 1-3 pN, and unfolding rates 15-40 pN. The obtained unfolding rates show different force sensitivities at forces below and above ∼27 pN, which determines a free-energy landscape with two energy barriers. Our results indicate that the free-energy landscape of small globule proteins have general Bactrian camel shape, and large contact order of the native state produces a high barrier dominate at low forces.

Clinical value of serum miR-92 and miR-122 expression level combined with pulmonary ultrasound score in the prognosis of neonatal acute respiratory distress syndrome.

PURPOSE: To explore the value of the expression of serum miR-92 and miR-122 combined with lung ultrasound score (LUS) in the prognosis of neonatal acute respiratory distress syndrome (ARDS). PATIENTS AND METHODS: This study involved 148 neonatal ARDS cases from January 2018 to October 2021, of which 77 children were discharged from hospital and 31 died. The children with ARDS were classified according to disease severity based on X-ray examination as mild (n = 69 cases) and severe (n = 39 cases). The expression of serum miR-92 and miR-122 was detected by real-time fluorescence quantitative PCR and the LUS score was recorded. The data were subjected to ROC curve analysis and Pearson correlation analysis. RESULTS: The expression of serum miR-92, miR-122, and LUS score in the patients that died were significantly higher than in those who survived (p < 0.05). These indicators were also significantly higher in the severe disease group compared to the mild disease group (p < 0.05). ROC curve showed that serum miR-92 and miR-122 combined with the LUS score had the largest area under the curve (0.920, 95% CI: 0.860-0.977) for predicting death, with a sensitivity and specificity of 92.0% and 87.0%, respectively. Pearson correlation analysis showed that the expression levels of serum miR-92 and miR-122 were positively correlated with the LUS score (all p < 0.01). CONCLUSIONS: The increased expression of serum miR-92 and miR-122 is related to the severity and prognosis of children with ARDS, combined with the LUS score are of value to predict the prognosis of children with ARDS.

Head-to-head comparison of [68 Ga]Ga-P16-093 and [68 Ga]Ga-PSMA-617 in dynamic PET/CT evaluation of the same group of recurrent prostate cancer patients.

PURPOSE: This study was prospectively designed to evaluate the early dynamic organ distribution and tumor detection capability of [68 Ga]Ga-P16-093, which was compared with [68 Ga]Ga-PSMA-617 in the same group of recurrent prostate cancer patients. METHODS: Twenty patients with recurrent prostate cancer were enrolled. In 2 consecutive days, each patient underwent a 60-min dynamic PET/CT scan after intravenous administration of 148-185 MBq (4-5 mCi) [68 Ga]Ga-P16-093 and [68 Ga]Ga-PSMA-617, respectively. Following a low-dose CT scan, serial dynamic PET scans were performed from head to proximal thigh at 9 time points (30 s/bed at 4, 7, 10, 13, and 16 min; 1 min/bed at 20, 30, and 45 min; and 2 min/bed at 60 min). Standardized uptake values were measured for semi-quantitative comparison. RESULTS: [68 Ga]Ga-P16-093 PET/CT revealed a significantly higher tumor uptake at 4 min (SUVmax 7.88 ± 5.26 vs. 6.01 ± 3.88, P < 0.001), less blood pool retention at 4 min (SUVmean 5.12 ± 1.16 vs. 6.14 ± 0.98, P < 0.001), and lower bladder accumulation at 60 min (SUVmean 31.33 ± 27.47 vs. 48.74 ± 34.01, P = 0.042) than [68 Ga]Ga-PSMA-617 scan. Significantly higher [68 Ga]Ga-P16-093 uptakes were also observed in the parotid gland, liver, spleen, and kidney. Besides, [68 Ga]Ga-P16-093 exhibited a better detectability of tumor than [68 Ga]Ga-PSMA-617 (366 vs. 321, P = 0.009). CONCLUSIONS: [68 Ga]Ga-P16-093 showed advantages over [68 Ga]Ga-PSMA-617 with higher tumor uptakes, tumor-to-blood pool ratio and detection capability, less blood pool, and bladder accumulation in recurrent prostate cancer patients. TRIAL REGISTRATION: [68 Ga]Ga-P16-093 and [68 Ga]Ga-PSMA-617 PET/CT Imaging in the Same Group of Prostate Cancer Patients (NCT04796467, Registered 12 March 2021, retrospectively registered) URL of registry: https://clinicaltrials.gov/ct2/show/NCT04796467.

Kit-based preparation of [68Ga]Ga-P16-093 (PSMA-093) using different commercial 68Ge/68Ga generators.

PURPOSE: Prostate-specific membrane antigen (PSMA) is an important biomarker for molecular imaging and a target for radionuclide therapy of prostate cancer. Recently, U.S. Food and Drug Administration (FDA) has approved [68Ga]Ga-PSMA-11 as a PSMA-targeted positron emission tomography (PET) imaging agent for the diagnosis of prostate cancer. As an alternative PSMA imaging agent, [68Ga]Ga-P16-093 ([68Ga]Ga-PSMA-093) showed excellent blood clearance and rapid tumor uptake, desirable in vivo properties for avidly detecting primary tumor and metastatic lesions in patients. To improve the availability and test the robustness of radiolabeling reaction, eluents of 68Ga/HCl from different sources of generators were evaluated. PROCEDURES: Commercially available 68Ge/68Ga generators from Eckert & Ziegler, ITG and iThemba were eluted with varying molarities of hydrochloric acid (0.05-0.6 M, as recommended by each company) and reacted with P16-093 kits. Radiolabeling yields, in vitro stabilities, in vitro cell uptakes and drug release criteria of different preparations were investigated. PET/computed tomography (CT) imaging of prostate cancer patients with [68Ga]Ga-P16-093 produced by using different sources of 68Ga were performed. RESULTS: Optimized P16-093 kit containing 15 µg of P16-093 (precursor) and 68 mg of sodium acetate trihydrate (buffer), a formulation previously tested in humans, was successfully labeled with eluents from Eckert & Ziegler, ITG and iThemba's generators. In vitro cell uptake studies showed that [68Ga]Ga-P16-093, formulated with ITG and iThemba's generators, exhibited equivalent PSMA-specific uptakes. Clinical studies in prostate cancer patients exhibited exceedingly comparable maximum standardized uptake value (SUVmax) for each lesion regardless of source of the generator used in preparation. CONCLUSION: Using different vendors' generator and lyophilized P16-093 kits, [68Ga]Ga-P16-093 could be conveniently and reliably prepared by a simple one-step reaction with excellent yields. Clinically useful doses of [68Ga]Ga-P16-093 imaging tracer could be made available using different 68Ge/68Ga generators.

Evaluating [68Ga]Ga-p14-032 as a Novel PET Tracer for Diagnosis Cerebral Amyloid Angiopathy.

Objective: We aimed to investigate the distribution of [68Ga]Ga-p14-032, a novel PET ligand that binds to vascular amyloid, in patients diagnosed clinically with probable cerebral amyloid angiopathy (CAA) compared with patients with Alzheimer's disease (AD) and normal controls (NC). Methods: This longitudinal cohort study was composed of 10 subjects (three probable CAA patients, two AD patients, five NC subjects), recruited from a clinic in China. CAA patients had a history of lobar intracerebral hemorrhage (ICH) and met modified Boston criteria for probable CAA. All participants were aged at least 55 years and underwent [68Ga] Ga-p14-032 PET/CT or/and PET/MRI, and the Montreal Cognitive Assessment on initial assessment. Demographics were measured at baseline (diabetes, hypertension, hypercholesterolemia, ischemic stroke, and ICH). Two PET imaging experts reviewed the PET images with cortical standardized uptake value ratio (SUVr) displayed on a color scale and visually classified the images as positive or negative. The mean of SUVr was calculated using the pons as reference. Results: In CAA patients, PET scans were positive in regions with higher numbers of CMBs. No significant signal was seen in AD subjects or controls. The relative [68Ga]Ga-p14-032 retention in the cortex was stronger in patients with CAA than AD and NC (median SUVr 2.68 ± 1.53 vs. 1.77 ± 0.08 and 0.83 ± 0.24). Conclusions: Our results provide early evidence that the [68Ga] Ga-p14-032 PET probe binds preferentially to vascular amyloid and may be a useful tracer for diagnosing CAA.

68Ga-labelled-exendin-4: New GLP1R targeting agents for imaging pancreatic ß-cell and insulinoma.

OBJECTIVE: Glucagon-like peptide-1 receptor (GLP1R) specifically expressed on the surface of pancreatic ß-cells and insulinoma, is a potential biomarker for imaging ß-cell mass (BCM). In this study, two new 68Ga-labelled GLP1R targeting agents were prepared and their biological properties for imaging BCM and insulinoma were evaluated. METHODS: [68Ga]Ga-HBED-CC-MAL-Cys39-exendin-4 ([68Ga]Ga-4) and its dimer ([68Ga]Ga-5) were synthesized from corresponding precursors. Cell uptake studies were evaluated in INS-1 cells. Biodistribution and microPET studies were performed in male normal Sprague-Dawley rats, diabetic rats and insulinoma xenograft NOD/SCID mice. RESULTS: [68Ga]Ga-4 and [68Ga]Ga-5 were efficiently radiolabelled by a simple one-step reaction without purification leading to high radiochemical yields and radiochemical purities (both >95%, decay corrected, n = 6, molar activity 15 GBq/µmol). They both showed excellent stability (~95%) in phosphate-buffered saline, pH 7.4, and in rat serum (~90%) for 2 h. Biodistribution studies and small animal PET/CT imaging showed that [68Ga]Ga-4 displayed specific uptake in rat pancreas and mouse insulinoma, and a reduced uptake in the pancreas of diabetic rat was observed (~62% reduction). Notably, it exhibited a rapid time-to-peak pancreatic uptake (0.96 ± 0.19%ID/g in 15 min) and fast clearance from the kidney (42% clearance in 30 min). Results suggested a favorable in vivo kinetics for human imaging studies. CONCLUSIONS: [68Ga]Ga-4 targeting GLP1R of pancreatic ß-cells may be a potentially useful PET agent and a suitable candidate for further structural modification studies. This agent has demonstrated several advantages, rapid time-to-peak pancreatic uptake and faster clearance from the kidney, factors may enhance diagnosis of diabetes and insulinoma.

SpyTag/SpyCatcher tether as a fingerprint and force marker in single-molecule force spectroscopy experiments.

Single molecule force spectroscopy has emerged as a powerful tool to study protein folding dynamics, ligand-receptor interactions, and various mechanobiological processes. High force precision does not necessarily lead to high force accuracy, as the uncertainties in calibration can bring serious systematic errors. In the case of magnetic tweezers, accurate determination of the applied forces for short biomolecular tethers, by measuring thermal fluctuations of inhomogeneous magnetic beads, remains difficult. Here we address this challenge by showing that the SpyTag/SpyCatcher complex is not only a convenient and genetically encodable covalent linker but also an ideal molecular fingerprint and force marker in single molecule force spectroscopy experiments. By stretching the N-termini of both SpyCatcher and SpyTag, the complex unfolds locally up to the isopeptide bond position in an unzipping geometry, resulting in equilibrium transitions at ∼30 pN with step sizes of ∼3.4 nm. This mechanical feature can be used as the fingerprint to identify single-molecular events. Moreover, the transitions occur with a fast exchange rate and in a narrow force range. Therefore, the real applied forces can be determined accurately based on the force-dependent transitions. The equilibrium forces are insensitive to buffer conditions and temperature, making the calibration applicable to many complicated experimental systems. We provide an example to calibrate protein unfolding forces using this force marker and expect that this method can greatly simplify force calibration in single-molecule force spectroscopy experiments and improve the force accuracy.

Development and validation of a kit formulation of [68Ga]Ga-P15-041 as a bone imaging agent.

One of the commonly performed studies in nuclear medicine are bone scans with [99mTc]Tc-methylene diphosphonate (MDP) for detecting various bone lesions, including cancer metastasis. The recent emergence of commercially available 68Ge/68Ga radionuclide generators makes it possible to provide 68Ga-labelled bisphosphonates as positron emission tomography (PET) tracers for bone imaging. Preliminary human studies suggested that [68Ga]Ga-HBED-CC-BP ([68Ga]Ga-P15-041) in conjunction with PET/computed tomography (CT) showed accumulation in known bone lesions, fast clearance from blood and soft tissue, and an ability to provide high contrast images. A simple and efficient lyophilized P15-041 kit formulation for the rapid production of [68Ga]Ga-P15-041 with excellent radiochemical purity (RCP) under ambient temperature without the need for purification is described. It is demonstrated that clinical doses of [68Ga]Ga-P15-041 can be prepared manually within minutes with an excellent purity (> 90%) and readily meet the dose release criteria. When [68Ga]Ga-P15-041 was evaluated in a patient with cancer, the imaging agent clearly showed accumulations in multiple lesions. In conclusion, [68Ga]Ga-P15-041, prepared by a lyophilized kit, might be an excellent bone imaging agent for widespread clinical application.

Exploring the Key Factors of Shared Decision-Making Through an Influential Network Relation Map: The Orthopedic Nurse's Perspective.

BACKGROUND: Few studies have used quantitative methods to explore the key factors affecting shared decision-making (SDM) in nursing decision-making from the perspective of orthopedic nurses. PURPOSE: To understand the intercorrelations among shared decision-making questionnaire-nurse (SDM-Q-NUR) factors and identify key factors for clinical nursing care decisions in orthopedics. METHODS: In May 2021, this study investigated the interdependence of the SDM-Q-NUR scale and developed an influential network-relation map (INRM) from the clinical experience of 13 trained orthopedic nurses using the Decision-making Trial and Evaluation Laboratory method. RESULTS: The INRM results showed that the nine criteria corresponded to three stages: preparation, discussion, and decision. "I helped my patient or patient's family understand all the information" (C 5) and "I wanted to know from my patient or patient's family how they want to be involved in making the nursing care decision" (C 2) are the main key factors for the beginning of nursing decision. In the discussion and decision stages, the corresponding key factors are "I made it clear to my patient or patient's family that a nursing care decision needs to be made" (C 1) and "I asked my patient or patient's family which nursing care option they prefer" (C 6). The result's statistical significance confidence and gap error were 98.106% and 1.894%, respectively. CONCLUSIONS: When making nursing decisions with patients, orthopedic nurses need to have detailed information about how patients are involved in SDM and all relevant information. Nurses should also inform patients and their families regarding the purpose of the discussion, namely, to help one understand the content, advantages, and disadvantages of the nursing care options, and finally, make a decision.

Two energy barriers and a transient intermediate state determine the unfolding and folding dynamics of cold shock protein.

Cold shock protein (Csp) is a typical two-state folding model protein which has been widely studied by biochemistry and single molecule techniques. Recently two-state property of Csp was confirmed by atomic force microscopy (AFM) through direct pulling measurement, while several long-lifetime intermediate states were found by force-clamp AFM. We systematically studied force-dependent folding and unfolding dynamics of Csp using magnetic tweezers with intrinsic constant force capability. Here we report that Csp mostly folds and unfolds with a single step over force range from 5 pN to 50 pN, and the unfolding rates show different force sensitivities at forces below and above ~8 pN, which determines a free energy landscape with two barriers and a transient intermediate state between them along one transition pathway. Our results provide a new insight on protein folding mechanism of two-state proteins.

Protein folding mechanism revealed by single-molecule force spectroscopy experiments.

Force spectroscopy experiments use mechanical force as a control factor to regulate the folding and unfolding process of proteins. Atomic force microscopy has been widely used to study the mechanical stability of proteins, and obtained unfolding forces and unfolding distance of different proteins, while recently, more low force folding and unfolding measurements were done by optical tweezers and magnetic tweezers. Due to the relatively small distortion of the free energy landscape, low force measurements give the free energy landscape information over bigger conformational space. In this review, we summarize the results of force spectroscopy experiments on different proteins. The unfolding distance obtained at high forces by atomic force microscopy are mostly smaller than 2 nm, while the unfolding distances at low forces distribute over a larger range: from a negative value to more than 6 nm. The sizes of the transition states at low force are ~4 nm for most compact two-state globular proteins, which indicates that this transition state might be the general free energy barrier separating the unfolded state and the theoretically predicated molten globule state. Up to now, only a limited number of proteins has been studied at low forces. We expect that more and more proteins with different conformations will be studied at low forces to reveal the general protein folding mechanism.

Hidden Intermediate State and Second Pathway Determining Folding and Unfolding Dynamics of GB1 Protein at Low Forces.

Atomic force microscopy experiments found that GB1, a typical two-state model protein used for study of folding and unfolding dynamics, can sustain forces of more than 100 pN, but its response to low forces still remains unclear. Using ultrastable magnetic tweezers, we discovered that GB1 has an unexpected nonmonotonic force-dependent unfolding rate at 5-160 pN, from which a free energy landscape with two main barriers and a hidden intermediate state was constructed. A model combining two separate models by Dudko et al. with two pathways between the native state and this intermediate state is proposed to rebuild the unfolding dynamics over the full experimental force range. One candidate of this transient intermediate state is the theoretically proposed molten globule state with a loosely collapsed conformation, which might exist universally in the folding and unfolding processes of two-state proteins.