Distribution of membrane trafficking system components across ciliate diversity highlights heterogenous organelle-associated machinery.

The ciliate phylum is a group of protists noted for their unusual membrane trafficking system and apparent environmental ubiquity; as highly successful microbial predators, they are found in all manner of environments and the ability for specific species to adapt to extremely challenging conditions makes them valued as bioindicators. Ciliates have also been used for many years as cell biological models because of their large cell size and ease of culturing, and for many fundamental cell structures, particularly membrane-bound organelles, ciliates were some of the earliest organisms in which these were observed via microscopy. In this study, we carried out a comparative genomic survey of selected membrane trafficking proteins in a pan-ciliate transcriptome and genome dataset. We observed considerable loss of membrane trafficking system (MTS) proteins that would indicate a loss of machinery that is generally conserved across eukaryotic diversity, even after controlling for potentially incomplete genome representation. In particular, the complete DSL1 complex was missing in all surveyed ciliates. This protein complex has been shown as involved in peroxisome biogenesis in some model systems, and a paucity of DSL1 components has been indicative of degenerate peroxisome. However, Tetrahymena thermophila (formerly Tetrahymena pyroformis) was one of the original models for visualizing peroxisomes. Conversely, the AP3 complex essential for mucocyst maturation in T. thermophila, is poorly conserved despite the presence of secretory lysosome-related organelles across ciliate diversity. We discuss potential resolutions for these apparent paradoxes in the context of the heterogenous distribution of MTS machinery across the diversity of ciliates.

Detection of HER2 expression using 99mTc-NM-02 nanobody in patients with breast cancer: a non-randomized, non-blinded clinical trial.

BACKGROUND: 99mTc radiolabeled nanobody NM-02 (99mTc-NM-02) is a novel single photon emission computed tomography (SPECT) probe with a high affinity and specificity for human epidermal growth factor receptor 2 (HER2). In this study, a clinical imaging trial was conducted to investigate the relationship between 99mTc-NM-02 uptake and HER2 expression in patients with breast cancer. METHODS: Thirty patients with pathologically confirmed breast cancer were recruited and imaged with both 99mTc-NM-02 SPECT/computed tomography (CT) and 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/CT. According to the treatment conditions before recruitment, patients were divided into two groups, the newly diagnosed group (n = 24) and the treated group (n = 6). The maximal standard uptake value (SUVmax) of 18F-FDG and SUVmax and mean SUV (SUVmean) of 99mTc-NM-02 in the lesions were determined to analyze the relationship with HER2 expression. RESULTS: No meaningful relationship was observed between 18F-FDG uptake and HER2 expression in 30 patients with breast cancer. 99mTc-NM-02 uptake was positively correlated with HER2 expression in the newly diagnosed group, but no correlation was observed in the treated group. 99mTc-NM-02 uptake in HER2-positive lesions was lower in those with effective HER2-targeted therapy compared with the newly diagnosed group. 99mTc-NM-02 SPECT/CT detected brain and bone metastases of breast cancer with a different imaging pattern from 18F-FDG PET/CT. 99mTc-NM-02 showed no non-specific uptake in inflamed tissues and revealed intra- and intertumoral HER2 heterogeneity by SPECT/CT imaging in 9 of the 30 patients with breast cancer. CONCLUSIONS: 99mTc-NM-02 SPECT/CT has the potential for visualizing whole-body HER2 overexpression in untreated patients, making it a promising method for HER2 assessment in patients with breast cancer. TRIAL REGISTRATION: NCT04674722, Date of registration: December 19, 2020.

Self-supervised learning for denoising of multidimensional MRI data.

PURPOSE: To develop a fast denoising framework for high-dimensional MRI data based on a self-supervised learning scheme, which does not require ground truth clean image. THEORY AND METHODS: Quantitative MRI faces limitations in SNR, because the variation of signal amplitude in a large set of images is the key mechanism for quantification. In addition, the complex non-linear signal models make the fitting process vulnerable to noise. To address these issues, we propose a fast deep-learning framework for denoising, which efficiently exploits the redundancy in multidimensional MRI data. A self-supervised model was designed to use only noisy images for training, bypassing the challenge of clean data paucity in clinical practice. For validation, we used two different datasets of simulated magnetization transfer contrast MR fingerprinting (MTC-MRF) dataset and in vivo DWI image dataset to show the generalizability. RESULTS: The proposed method drastically improved denoising performance in the presence of mild-to-severe noise regardless of noise distributions compared to previous methods of the BM3D, tMPPCA, and Patch2self. The improvements were even pronounced in the following quantification results from the denoised images. CONCLUSION: The proposed MD-S2S (Multidimensional-Self2Self) denoising technique could be further applied to various multi-dimensional MRI data and improve the quantification accuracy of tissue parameter maps.

Pre-clinical evaluation of 99mTc-labeled chalcone derivative for amyloid-ß imaging post-head trauma.

Aß42 plaque formation is one of the preliminary pathologic events that occur post traumatic brain injury (TBI) which is also among the most noteworthy hallmarks of AD. Their pre symptomatic detection is therefore vital for better disease management. Chalcone-picolinic acid chelator derivative, 6-({[(6-carboxypyridin-2-yl)methyl](2-{4-[(2E)-3-[4-(dimethyl amino)phenyl]prop-2-enoyl]phenoxy}ethyl)amino}methyl)pyridine-2-carboxylic acid, Py-chal was synthesized to selectively identify amyloid plaques formed post head trauma using SPECT imaging by stable complexation to 99mTc with > 97% efficiency without compromising amyloid specificity. The binding potential of the Py-chal ligand to amyloid plaques remained high as confirmed by in vitro binding assay and photophysical spectra. Further, the Py-chal complex stained amyloid aggregates in the brain sections of rmTBI mice model. In vivo scintigraphy in TBI mice model displayed high uptake followed by high retention while the healthy rabbits displayed higher brain uptake followed by a rapid washout attributed to absence of amyloid plaques. Higher uptake in brain of TBI model was also confirmed by ex vivo biodistribution analysis wherein brain uptake of 3.38 ± 0.2% ID/g at 2 min p.i. was observed for TBI mice model. This was followed by prolonged retention and more than twofold higher activity as compared to sham mice brain. This preliminary data suggests the specificity of the radiotracer for amyloid detection post head trauma and applicability of 99mTc labeled Py-chal complex for TBI-induced ß-amyloid SPECT imaging.

Comparison of Cardiac Activated Fibroblast Imaging and Magnetic Resonance Imaging in Patients with COVID-19-Related Myocarditis.

Background: This study aimed to explore the association between cardiac fibroblast activation and cardiac magnetic resonance (CMR) imaging parameters in patients with myocarditis following infection with coronavirus 2019 (COVID-19). Methods: In this prospective study, four patients with COVID-19-related myocarditis underwent 99mTc-labeled-hydrazinonicotinamide-fibroblast activation protein inhibitor-04 (99mTc-HFAPi) single photon emission computed tomography/computed tomography (SPECT/CT) and CMR imaging. Segmental 99mTc-HFAPi activity was quantified as the percentage of average segmental myocardial count × global left ventricular target-to-background ratio. T1/T2 values, extracellular volume (ECV), and late gadolinium enhancement (LGE) were analyzed by CMR. The consistency between myocardial 99mTc-HFAPi activity and CMR parameters was explored. Results: In patients with myocarditis, the proportion of segments with abnormal 99mTc-HFAPi activity was significantly higher than in those with abnormal LGE (81.25% vs. 60.93%, p = 0.011), abnormal T2 (81.25% vs. 50.00%, p < 0.001), and abnormal ECV (81.25% vs. 59.38%, p = 0.007); however, they were similar in those with abnormal native T1 (81.25% vs. 73.43%, p = 0.291). Meanwhile, 99mTc-HFAPi imaging exhibited good consistency with native T1 (kappa = 0.69). Conclusions: Increased cardiac 99mTc-HFAPi activity is present in COVID-19-related myocarditis, which is correlated with the native T1 values in CMR.

Sentinel lymph node detection in thyroid carcinoma using [68Ga]Ga-tilmanocept PET/CT: a proof-of-concept study.

PURPOSE: Sentinel lymph node (SLN) biopsy is rarely used for thyroid carcinoma staging. This is due to challenges associated with conventional Tc-99m-labeled tracers, often producing a large hotspot at the injection site, potentially hiding nearby SLNs (shine-through effect). The aim of this study was to demonstrate the feasibility and effectiveness of SLN visualization using the new PET tracer [68Ga]Ga-tilmanocept. METHODS: Patients with thyroid carcinoma underwent ultrasound-guided peritumoral injection of [68Ga]Ga-tilmanocept and ICG-[99mTc]Tc-nanocolloid. [68Ga]Ga-tilmanocept PET/CT scans were conducted at 15 min and 60 min post-injection to visualize the SLNs. SLN biopsy was performed using ICG-[99mTc]TC-nanocolloid for intraoperative identification. The corresponding lymph node level was resected for reference. RESULTS: Seven differentiated thyroid carcinoma (DTC) and 3 medullary thyroid carcinoma (MTC) patients were included, of which 6 were clinically node-negative. The median number of SLNs detected on [68Ga]Ga-tilmanocept PET/CT and resected was 3 (range 1-4) and 3 (range 1-5), respectively. Eight SLNs were found on PET/CT in the central compartment and 19 in the lateral compartment. The SLN procedure detected (micro)metastases in all patients except one. Seventeen of 27 pathologically assessed SLNs were positive, 8 negative, and 2 did not contain lymph node tissue, which led to upstaging in 5 out of 6 clinically node-negative patients. CONCLUSIONS: [68Ga]Ga-tilmanocept PET/CT identified SLNs in all patients, mainly in the lateral neck. The SLNs were successfully surgically detected and resected using ICG-[99mTc]Tc-nanocolloid. This technique has the potential to improve neck staging, enabling more personalized treatment of thyroid cancer according to the lymph node status. TRIAL REGISTRATION: 2021-002470-42 (EudraCT).

Molecular genetics, therapeutics and RET inhibitor resistance for medullary thyroid carcinoma and future perspectives.

Medullary thyroid carcinoma (MTC) is a rare type of thyroid malignancy that accounts for approximately 1-2% of all thyroid cancers (TCs). MTC include hereditary and sporadic cases, the former derived from a germline mutation of rearrangement during transfection (RET) proto-oncogene, whereas somatic RET mutations are frequently present in the latter. Surgery is the standard treatment for early stage MTC, and the 10-year survival rate of early MTC is over 80%. While for metastatic MTC, chemotherapy showing low response rate, and there was a lack of effective systemic therapies in the past. Due to the high risk (ca. 15-20%) of distant metastasis and limited systemic therapies, the 10-year survival rate of patients with advanced MTC was only 10-40% from the time of first metastasis. Over the past decade, targeted therapy for RET has developed rapidly, bringing hopes to patients with advanced and progressive MTC. Two multi-kinase inhibitors (MKIs) including Cabozantinib and Vandetanib have been shown to increase progression-free survival (PFS) for patients with metastatic MTC and have been approved as choices of first-line treatment. However, these MKIs have not prolonged overall survival (OS) and their utility is limited due to high rates of off-target toxicities. Recently, new generation TKIs, including Selpercatinib and Pralsetinib, have demonstrated highly selective efficacy against RET and more favorable side effect profiles, and gained approval as second-line treatment options. Despite the ongoing development of RET inhibitors, the management of advanced and progressive MTC remains challenging, drug resistance remains the main reason for treatment failure, and the mechanisms are still unclear. Besides, new promising therapeutic approaches, such as novel drug combinations and next generation RET inhibitors are under development. Herein, we overview the pathogenesis, molecular genetics and current management approaches of MTC, and focus on the recent advances of RET inhibitors, summarize the current situation and unmet needs of these RET inhibitors in MTC, and provide an overview of novel strategies for optimizing therapeutic effects.

Synthesis and Evaluation of 99mTc-Labeled DPro-Gly-Containing Tracers Targeting PSMA.

The specific expression of prostate-specific membrane antigen (PSMA) makes it an ideal target for the diagnosis and treatment of prostate cancer. Currently, many 99mTc-labeled PSMA-targeted tracers have been developed. However, the high renal uptake of these 99mTc-labeled tracers is a common problem that limits their clinical application. In this work, the ligand (EUKPG) using DPro-Gly as the linker was synthesized and three 99mTc-labeled complexes ([99mTc]Tc-EUKPG-EDDA, [99mTc]Tc-EUKPG-TPPTS, [99mTc]Tc-EUKPG-TPPMS) with different coligands were prepared and evaluated. Among them, [99mTc]Tc-EUKPG-EDDA showed the most favorable pharmacokinetic properties, with significantly reduced uptake in the kidney (14.04 ± 0.23% ID/g), rapid clearance and low uptake in nontarget organs, thus making it to exhibit high tumor-to-background ratios (tumor/blood: 7.47, tumor/muscle: 12.65). Affinity studies have shown that it has high specificity for PSMA both in vivo and in vitro. Therefore, [99mTc]Tc-EUKPG-EDDA has great potential as a promising molecular tracer to target PSMA for tumor imaging.

Preparation and Evaluation of a Novel 99mTc-Labeled Niraparib Isonitrile Complex as a Potential PARP-1 Imaging Agent.

Poly ADP-ribose polymerase (PARP) plays an important role in the DNA repair process and has become an attractive target for cancer therapy in recent years. Given that niraparib has good clinical efficacy as a PARP inhibitor, this study aimed to develop radiolabeled niraparib derivatives for tumor imaging to detect PARP expression and improve the accuracy of stratified patient therapy. The niraparib isonitrile derivative (CNPN) was designed, synthesized, and radiolabeled to obtain the [99mTc]Tc-CNPN complex with high radiochemical purity (>95%). It was lipophilic and stable in vitro. In HeLa cell experiments, the uptake of [99mTc]Tc-CNPN was effectively inhibited by the ligand CNPN, indicating the binding affinity for PARP. According to the biodistribution studies of HeLa tumor-bearing mice, [99mTc]Tc-CNPN has moderate tumor uptake and can be effectively inhibited, demonstrating its specificity for targeting PARP. The SPECT imaging results showed that [99mTc]Tc-CNPN had tumor uptake at 2 h postinjection. All of the results of this study indicated that [99mTc]Tc-CNPN is a promising tumor imaging agent that targets PARP.

Synthesis and Preliminary Study of 99mTc-Labeled HYNIC-FAPi for Imaging of Fibroblast Activation Proteins in Tumors.

Fibroblast activation protein (FAP) is an emerging target for cancer diagnosis. Different types of FAP inhibitor (FAPI)-based radiotracers have been developed and applied for tumor imaging. However, few FAPI tracers for single photon emission computed tomography (SPECT) imaging have been reported. SPECT imaging is less expensive and more widely distributed than positron emission tomography (PET), and thus, 99mTc-labeled FAPIs would be more available to patients in developing regions. Herein, we developed a FAPI-04-derived radiotracer, HYNIC-FAPi-04 (HFAPi), for SPECT imaging. 99mTc-HFAPi, with a radiochemical purity of >98%, was prepared using a kit formula within 30 min. The specificity of 99mTc-HFAPi for FAP was validated by a cell binding assay in vitro and SPECT/CT imaging in vivo. The binding affinity (Kd value) of 99mTc-HFAPi for human FAP and murine FAP was 4.49 and 2.07 nmol/L, respectively. SPECT/CT imaging in HT1080-hFAP tumor-bearing mice showed the specific FAP targeting ability of 99mTc-HFAPi in vivo. In U87MG tumor-bearing mice, 99mTc-HFAPi had a higher tumor uptake compared with that of HT1080-hFAP and 4T1-mFAP tumor models. Interestingly, 99mTc-HFAPi showed a relatively high uptake in some murine joints. 99mTc-HFAPi accumulated in tumor lesions with a high tumor-to-background ratio. A preliminary clinical study was also performed in breast cancer patients. Additionally, 99mTc-HFAPi exhibited an advantage over 18F-FDG in the detection of lymph node metastatic lesions in breast cancer patients, which is helpful in improving treatment strategies. In short, 99mTc-HFAPi showed excellent affinity and specificity for FAP and is a promising SPECT radiotracer for (re)staging and treatment planning of breast cancers.

Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the 99mTc-Based Imaging of HER2-Expressing Malignant Tumors.

HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three 99mTc-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (G3C), (Gly-Gly-Gly-Ser)3-Cys ((G3S)3C), or Glu-Glu-Glu-Cys (E3C) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with 99mTc(CO)3 using the (HE)3-tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50-80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5-3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of 99mTc-labeled DARPin G3 variants to HER2 in vitro; however, [99mTc]Tc-G3-(G3S)3C-HYNIC had the highest affinity. Comparative biodistribution of [99mTc]Tc-G3-G3C-HYNIC, [99mTc]Tc-G3-(G3S)3C-HYNIC, [99mTc]Tc-G3-E3C-HYNIC, and [99mTc]Tc-(HE)3-G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [99mTc]Tc-G3-E3C-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [99mTc]Tc-G3-(G3S)3C-HYNIC and [99mTc]Tc-(HE)3-G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [99mTc]Tc-G3-(G3S)3C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [99mTc]Tc-(HE)3-G3. Radiolabeling of DARPin G3-HYNIC conjugates with 99mTc provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the 99mTc-tricarbonyl-labeled DARPin G3. At this stage, [99mTc]Tc-(HE)3-G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.

Synthesis and Evaluation of Novel 99mTc-Labeled FGFR2-Targeting Peptides.

To develop radiolabeled FGFR2-targeting probes for visualizing fibroblast growth factor receptor (FGFR) expression levels in the tumor microenvironment, four novel 99mTc-labeled FGFR2-targeting peptides ([99mTc]Tc-FGFR2-1, [99mTc]Tc-FGFR2-2, [99mTc]Tc-FGFR2-3, and [99mTc]Tc-FGFR2-4) with different amino acid linkers between the targeted peptide moiety and the 99mTc chelating group were designed and synthesized. The in vitro cellular inhibition, internalization, and efflux results demonstrated that the four 99mTc complexes exhibited FGFR2-specific binding and prolonged cellular retention in DU145 human prostate cancer cells, which indicated that modification from the glycine side (N-terminal) of CH02 was feasible. Among them, [99mTc]Tc-FGFR2-1 exhibited the highest in vitro cellular uptake and in vivo tumor uptake at 30 min postinjection, and tumor uptake could be significantly inhibited by the competitor CH02 (53% inhibited, p < 0.05), suggesting the tumor-specific targeting ability of [99mTc]Tc-FGFR2-1. The DU145-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 30 min postinjection of [99mTc]Tc-FGFR2-1, highlighting its potential as a SPECT imaging probe for tumor FGFR2 detection.

Preclinical Evaluation of 99mTc-MAG3-5-Fab Targeting TREM2 in Lung Cancer Mouse Models: A Comparison with 99mTc-MAG3-5-F(ab')2.

Triggering receptor expressed on myeloid cells-2 (TREM2), which is expressed on the surface of tumor-associated macrophages (TAMs), has been found to play a major role in the diagnosis and treatment of tumors. TREM2 expression is significantly upregulated in tumor tissues, and therefore, targeting TREM2 for tumor imaging may be of value. Previously, we performed TREM2 targeting imaging by using 68Ga-NOTA-COG1410 or a 124I-labeled monoclonal antibody (mAb) and F(ab')2 in mouse models of colon and gastric tumors. However, some of the shortcomings of these probes (i.e., the high uptake of 68Ga-NOTA-COG1410 in the liver, the difficulty of obtaining iodine-124, and the long half-life of iodine-124) have hindered their clinical use. Herein, we sought to synthesize novel molecular probes targeting TREM2 that are more conducive to clinical translation, eliminating the interference of isotope availability and in vivo probe biodistribution issues. Therefore, we established A549 cell lines with negative human TREM2 (hTREM2) expression (GFP tag; hTREM2- A549) or upregulated hTREM2 expression (GFP tag; hTREM2+ A549) using lentiviral transfection and confirmed these with Western blotting and immunocytochemistry. We then prepared a mouse anti-human TREM2 (5-mAb) by immunizing with the hTREM2 antigen. The antibody fragments 5-F(ab')2 and 5-Fab were prepared from 5-mAb, and 99mTc-MAG3-5-F(ab')2 and 99mTc-MAG3-5-Fab were then synthesized with excellent stability and specificity. 99mTc-MAG3-5-F(ab')2 had a slightly higher in vitro affinity than 99mTc-MAG3-5-Fab (Kd = 3.32 ± 0.05 nmol versus 4.62 ± 0.85 nmol). 99mTc-MAG3-5-F(ab')2 and 99mTc-MAG3-5-Fab both showed excellent specificity: after adding a 100-fold precursor, the two probes binding to the cells were almost blocked. In vivo pharmacokinetics showed that the distribution and elimination half-lives of 99mTc-MAG3-5-Fab (T1/2α = 1.25 ± 0.30 min and T1/2ß = 21.98 ± 2.80 min, respectively) were significantly reduced compared to those of 99mTc-MAG3-5-F(ab')2 (T1/2α = 2.64 ± 0.37 min and T1/2ß = 86.55 ± 26.86 min, respectively). In micro single-photon emission computed tomography/computed tomography (micro-SPECT/CT) imaging, the tumor was clearly displayed at 1 h after 99mTc-MAG3-5-Fab injection, while the blood background was extremely low at 3 h, and the probe was mainly excreted through the kidneys and biliary tract. 99mTc-MAG3-5-F(ab')2 uptake was also detected at the tumor site, although the blood background was consistently high. The biodistribution results were consistent with the micro-SPECT/CT imaging results. 99mTc-MAG3-5-Fab could clearly display hTREM2+ A549 tumors in a short time (1 h) with low uptake in nontumor organs and tissues and thus has clinical application prospects.

Performance and Sensitivity of [99mTc]Tc-sestamibi Compared with Positron Emission Tomography Radiotracers to Measure P-glycoprotein Function in the Kidneys and Liver.

P-glycoprotein (P-gp, encoded in humans by the ABCB1 gene and in rodents by the Abcb1a/b genes) is a membrane transporter that can restrict the intestinal absorption and tissue distribution of many drugs and may also contribute to renal and hepatobiliary drug excretion. The aim of this study was to compare the performance and sensitivity of currently available radiolabeled P-gp substrates for positron emission tomography (PET) with the single-photon emission computed tomography (SPECT) radiotracer [99mTc]Tc-sestamibi for measuring the P-gp function in the kidneys and liver. Wild-type, heterozygous (Abcb1a/b(+/-)), and homozygous (Abcb1a/b(-/-)) Abcb1a/b knockout mice were used as models of different P-gp abundance in excretory organs. Animals underwent either dynamic PET scans after intravenous injection of [11C]N-desmethyl-loperamide, (R)-[11C]verapamil, or [11C]metoclopramide or consecutive static SPECT scans after intravenous injection of [99mTc]Tc-sestamibi. P-gp in the kidneys and liver of the mouse models was analyzed with immunofluorescence labeling and Western blotting. In the kidneys, Abcb1a/b() mice had intermediate P-gp abundance compared with wild-type and Abcb1a/b(-/-) mice. Among the four tested radiotracers, renal clearance of radioactivity (CLurine,kidney) was significantly reduced (-83%) in Abcb1a/b(-/-) mice only for [99mTc]Tc-sestamibi. Biliary clearance of radioactivity (CLbile,liver) was significantly reduced in Abcb1a/b(-/-) mice for [11C]N-desmethyl-loperamide (-47%), [11C]metoclopramide (-25%), and [99mTc]Tc-sestamibi (-79%). However, in Abcb1a/b(+/-) mice, CLbile,liver was significantly reduced (-47%) only for [99mTc]Tc-sestamibi. Among the tested radiotracers, [99mTc]Tc-sestamibi performed best in measuring the P-gp function in the kidneys and liver. Owing to its widespread clinical availability, [99mTc]Tc-sestamibi represents a promising probe substrate to assess systemic P-gp-mediated drug-drug interactions and to measure renal and hepatic P-gp function under different (patho-)physiological conditions.

Noninvasive Evaluation of Tumoral PD-L1 Using a Novel 99mTc-Labeled Nanobody Tracer with Rapid Renal Clearance.

The expression level of PD-L1 in tumor tissue is considered one of the effective biomarkers to guide PD-1/PD-L1 therapy. Quantifying whole-body PD-L1 expression by SPECT imaging may help in selecting patients that potentially respond to PD-1/PD-L1 therapy. Nanobody is the smallest antibody fragment with antigen-binding ability that is well suited for radionuclide imaging. Nevertheless, high retention of radioactivity in the kidney may limit its clinical translation. The present study aimed to screen, design, and prepare a nanobody-based SPECT probe with rapid renal clearance to evaluate the PD-L1 expression level in vivo noninvasively. A phage library was constructed by immunizing alpaca with recombinant human PD-L1 protein, and 17 anti-PD-L1 nanobodies were screened by the phage display technique. After sequence alignment and flow cytometry analysis, APN09 was selected as the candidate nanobody, and a GGGC chelator was attached to its C-terminus for 99mTc labeling to prepare a SPECT imaging probe. The affinity and specificity of 99mTc-APN09 were evaluated by protein and cell-binding experiments, and SPECT imaging and biodistribution were performed in a mouse model with bilateral transplantation of A549 and A549PD-L1 tumors. The ability of 99mTc-APN09 to quantify the PD-L1 expression level in vivo was validated in tumor models with different PD-L1 expression levels. 99mTc-APN09 had a radiochemical purity higher than 99% and a binding equilibrium dissociation constant of 21.44 ± 1.65 nM with hPD-L1, showing high affinity. SPECT imaging results showed that 99mTc-APN09 could efficiently detect PD-L1-positive tumors within 0.5 h, and the quantitative results of SPECT were well correlated with the expression level of PD-L1 in cell lines. SPECT imaging and biodistribution results also showed that 99mTc-APN09 was rapidly cleared from the kidney in 2 h postinjection. 99mTc-APN09 was a simple and stable tool for visualizing PD-L1 expression in the whole body. In addition, due to its significant reduction in renal retention, it has better prospects for clinical translation.

Are 99mTC-Sestamibi Single Photon Emission Computed Tomography Scan Results Associated to the Parathyroid Cell Type in Primary Hyperparathyroidism?

INTRODUCTION: 99mTC-sestamibi scintigraphy (SPECT-CT) is a common imaging modality for parathyroid localization in primary hyperparathyroidism (PHPT). Prior studies have suggested that the cellular composition of parathyroid adenomas influences SPECT-CT imaging results. Other biochemical and anatomical factors may also play a role in false negative results. Therefore, after controlling for confounding variables, we sought to determine whether the histologic composition of parathyroid adenomas is associated to SPECT-CT results in patients with single gland disease causing PHPT. METHODS: A retrospective review of patients with PHPT due to confirmed single gland disease was performed over a 2-y period. A 1:1 propensity score matching was done between patients with positive and negative SPECT-CT results with regard to demographical, biochemical, and anatomical characteristics followed by blinded pathologic examination of cell composition in the matched pairs. RESULTS: Five hundred forty two patients underwent routine four gland exploration and 287 (53%) patients were found to have a single adenoma. Of those, 26% had a negative SPECT-CT result. There were significant differences between groups with regards to biochemical profile, gland location, and gland size. All of which became nonsignificant after propensity score matching. Adenomas were primarily composed of chief cells, with no difference between groups (95% versus 97%, P = 0.30). In the positive SPECT-CT group, chief cells were the dominant cell type in 68% of the cases, followed by mixed type (13%), oxyphil cells (12%), and clear cells (7%). This was similar to the negative SPECT-CT group (P = 0.22). CONCLUSIONS: While certain patient's clinical characteristics are associated with SPECT-CT imaging results, histologic cell type is not significantly associated.

Preparation and bioevaluation of a novel 99mTc-labelled propylene amine oxime (PnAO) containing two 4-methyl-2-nitroimidazole groups as a promising tumor hypoxia imaging agent.

Hypoxia is a common phenomenon in solid tumors, and its presence inhibits the efficacy of tumor chemotherapy and radiotherapy. Accurate measurement of hypoxia before tumor treatment is essential. Three propylene amine oxime (PnAO) derivatives with different substituents attached to 2-nitroimidazole were synthesized in the work, they are 3,3,9,9-tetramethyl-1,11-bis(4-bromo-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Br2P2), 3,3,9,9-tetramethyl-1,11-bis(4-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Me2P2) and 3,3,9,9-tetramethyl-1,11-bis(4,5-dimethyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (2Me2P2). The three compounds were radiolabeled with 99mTc to give three complexes([99mTc]Tc-Br2P2, [99mTc]Tc-Me2P2 and [99mTc]Tc-2Me2P2) with good in vitro stability. [99mTc]Tc-Me2P2 with a more suitable reduction potential had the highest hypoxic cellular uptake, compared with [99mTc]Tc-2P2 that have been previously reported, [99mTc]Tc-Br2P2 and [99mTc]Tc-2Me2P2. Biodistribution results in S180 tumor-bearing mice demonstrated that [99mTc]Tc-Me2P2 had the highest tumor-to-muscle (T/M) ratio (12.37 ± 1.16) at 2 h in the four complexes. Autoradiography and immunohistochemical staining results revealed that [99mTc]Tc-Me2P2 specifically targeted tumor hypoxic regions. The SPECT/CT imaging results showed that [99mTc]Tc-Me2P2 could target the tumor site. [99mTc]Tc-Me2P2 may become a potential hypoxia imaging agent.

Role of a novel endoplasmic reticulum-resident glycoprotein Mtc6/Ehg2 in high-pressure growth: stability of tryptophan permease Tat2 in Saccharomyces cerevisiae.

Deep-sea organisms are subjected to extreme conditions; therefore, understanding their adaptive strategies is crucial. We utilize Saccharomyces cerevisiae as a model to investigate pressure-dependent protein regulation and piezo-adaptation. Using yeast deletion library analysis, we identified 6 poorly characterized genes that are crucial for high-pressure growth, forming novel functional modules associated with cell growth. In this study, we aimed to unravel the molecular mechanisms of high-pressure adaptation in S. cerevisiae, focusing on the role of MTC6. MTC6, the gene encoding the novel glycoprotein Mtc6/Ehg2, was found to stabilize tryptophan permease Tat2, ensuring efficient tryptophan uptake and growth under high pressure at 25 MPa. The loss of MTC6 led to promoted vacuolar degradation of Tat2, depending on the Rsp5-Bul1 ubiquitin ligase complex. These findings enhance our understanding of deep-sea adaptations and stress biology, with broad implications for biotechnology, environmental microbiology, and evolutionary insights across species.

mTORC1-chaperonin CCT signaling regulates m6A RNA methylation to suppress autophagy.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the m6A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m6A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m6A RNA methylation and demonstrates their roles in suppressing autophagy.

LncRNA MTC-p65 Protein Mediates Skin Fibroblast Proliferation in Liaoning Cashmere Goat by Regulating the Nuclear Factor-Kappa B Signaling Pathway.

The aim of this study is to investigate the activation of NF-κB signaling pathway and the regulation of the expression of genes related to chorionic villus growth by the binding of LncRNA MTC (XLOC_005914) and p65 (transcription factor p65 [Capra hircus], XP_017898873.1). In addition, the regulation of LncRNA MTC and p65 binding on the proliferation of Liaoning Cashmere Goat skin fibroblasts is investigated. The upregulation of LncRNA MTC promoted the proliferation of skin fibroblasts, and the NF-κB signaling pathway played an important role in this process. Compared with the negative control (NC group), the expression of TNFα and NFKB2(NF-κB) genes was highly significantly up-regulated (P < 0.001), and NFKBIA(IκBɑ) genes were highly significantly down-regulated (P < 0.01) after LncRNA MTC overexpression (OE group). The expression levels of TNFα and NFκB-P-p65 proteins were upregulated in the OE group; NF-κB-p65 expression levels were upregulated in the nucleus, IκBα expression levels were downregulated in the cytoplasm, and P-IκBα expression levels were upregulated. LncRNA MTC and p65 proteins were co-localized in the cells. Meanwhile, LncRNA MTC and p65 protein showed significant nucleation in the OE group. RNA pull-down and LC-MS/MS verified that p65 protein was indeed an interacting protein of LncRNA MTC. LncRNA MTC binds to p65 protein, upregulates the expression of TNFα protein, nucleates p65 protein, and activates NF-κB signaling pathway to promote the proliferation of skin fibroblasts in Liaoning Cashmere Goat.