Verification of the effect of data-driven brain motion correction on PET imaging.

INTRODUCTION: Brain positron emission tomography/computed tomography (PET/CT) scans are useful for identifying the cause of dementia by evaluating glucose metabolism in the brain with F-18-fluorodeoxyglucose or Aß deposition with F-18-florbetaben. However, since imaging time ranges from 10 to 30 minutes, movements during the examination might result in image artifacts, which interfere with diagnosis. To solve this problem, data-driven brain motion correction (DDBMC) techniques are capable of performing motion corrected reconstruction using highly accurate motion estimates with high temporal resolution. In this study, we investigated the effectiveness of DDBMC techniques on PET/CT images using a Hoffman phantom, involving continuous rotational and tilting motion, each expanded up to approximately 20 degrees. MATERIALS AND METHODS: Listmode imaging was performed using a Hoffman phantom that reproduced rotational and tilting motions of the head. Brain motion correction processing was performed on the obtained data. Reconstructed images with and without brain motion correction processing were compared. Visual evaluations by a nuclear medicine specialist and quantitative parameters of images with correction and reference still images were compared. RESULTS: Normalized Mean Squared Error (NMSE) results demonstrated the effectiveness of DDBMC in compensating for rotational and tilting motions during PET imaging. In Cases 1 and 2 involving rotational motion, NMSE decreased from 0.15-0.2 to approximately 0.01 with DDBMC, indicating a substantial reduction in differences from the reference image across various brain regions. In the Structural Similarity Index (SSIM), DDBMC improved it to above 0.96 Contrast assessment revealed notable improvements with DDBMC. In continuous rotational motion, % contrast increased from 42.4% to 73.5%, In tilting motion, % contrast increased from 52.3% to 64.5%, eliminating significant differences from the static reference image. These findings underscore the efficacy of DDBMC in enhancing image contrast and minimizing motion induced variations across different motion scenarios. CONCLUSIONS: DDBMC processing can effectively compensate for continuous rotational and tilting motion of the head during PET, with motion angles of approximately 20 degrees. However, a significant limitation of this study is the exclusive validation of the proposed method using a Hoffman phantom; its applicability to the human brain has not been investigated. Further research involving human subjects is necessary to assess the generalizability and reliability of the presented motion correction technique in real clinical scenarios.

Timeframe Analysis of Novel Synthetic Cannabinoids Effects: A Study on Behavioral Response and Endogenous Cannabinoids Disruption.

This study investigates the impact of SCs consumption by assessing the effects of three novel synthetic cannabinoids (SCs); MDMB-CHMINACA, 5F-ADB-PINACA, and APICA post-drug treatment. SCs are known for their rapid onset (<1 min) and prolonged duration (≥5 h). Therefore, this research aimed to assess behavioral responses and their correlation with endocannabinoids (ECs) accumulation in the hippocampus, and EC's metabolic enzymes alteration at different timeframes (1-3-5-h) following drug administration. Different extents of locomotive disruption and sustained anxiety-like symptoms were observed throughout all-encompassing timeframes of drug administration. Notably, MDMB-CHMINACA induced significant memory impairment at 1 and 3 h. Elevated levels of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were detected 1 h post-MDMB-CHMINACA and 5F-ADB-PINACA administration. Reduced mRNA expression levels of fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL) (AEA and 2-AG degrading enzymes, respectively), and brain-derived neurotrophic factor (BDNF) occurred at 1 h, with FAAH levels remaining reduced at 3 h. These findings suggest a connection between increased EC content and decreased BDNF expression following SC exposure. Cognitive disruption, particularly motor coordination decline and progressive loss manifested in a time-dependent manner across all the analyzed SCs. Our study highlights the importance of adopting a temporal framework when assessing the effects of SCs.

[Surgical Therapy of Slipping Rib Syndrome].

We have operated on two cases of slipped ribs syndrome( SRS). Both patients were men in their 40s with a history of right thoracic trauma who were referred to us because of unexplained lower thoracic pain. The left rib was positive for hooking maneuver (lift test), and dynamic ultrasonography showed narrowing of the intercostal space, which led to the diagnosis of SRS. in the first case, the tip of the ninth rib cartilage was excised, and the ninth and tenth rib cartilages were sutured and fixed with No.2 fiber wire in two places with Z sutures. In the second case, the tip of the ninth rib cartilage was excised, the eighth and ninth ribs and the ninth and tenth ribs were fixed with No.2 fiber wire with Z sutures as in the first case, and a 0.7 mm thick poly-L-lactide (PLLA) plate was added between the eighth and tenth rib cartilages. In both cases, the postoperative course was good and the pain disappeared. SRS should be recognized as a disease and surgical treatment should be used as therapy.

[Thoracoscopic Right Upper Lobectomy in a Patient with a Right Aortic Arch:Report of a Case].

It has been reported that the recurrent nerve may not be recognized during mediastinal lymph node dissection in surgery for right upper lobe lung cancer associated with the right aortic arch. In the present case, a 66-year-old man underwent thoracoscopic right upper lobectomy for right upper lobe lung cancer associated with the right aortic arch. The gap between the superior vena cava and descending aortic arch was narrow, and the vagus nerve ran between the superior vena cava and the aorta. The recurrent laryngeal nerve was able to confirm. The vagus nerve ran the hilum of lung back side from arch of azygos vein on the peripheral side. The morphology of the right aortic arch varies from case to case, and if the vagus nerve and recurrent nerve are difficult to identify, the nerve may be hidden by the superior vena cava. If the nerve cannot be recognized, the space between the superior vena cava and the aorta should be dissected and confirmed. In addition, taping the vagus nerve and observing it from the caudal to the cephalic side may be useful for the recurrent nerve that turns to the mediastinum. In this case, magnification of the thoracoscope is extremely useful.

Median Nerve Neuropathy Caused by Persistent Median Artery Thrombosis.

Persistent median artery thrombosis mimicking carpal tunnel syndrome is rare. Here, we report the pathological, ultrasonography, and intraoperative findings of a case of persistent median artery thrombosis mimicking carpal tunnel syndrome. A 34-year-old man reported to our clinic with a complaint of numbness in his left thumb, index finger, and middle finger, which are innervated by the left median nerve. He also reported that he felt pain in his left wrist and distal forearm while working. Although findings of the usual provocative tests and nerve conduction studies were normal, ultrasonography revealed arterial thrombosis at the carpal tunnel level, whereas magnetic imaging showed persistent median artery thrombosis in the carpal tunnel. Three months after surgical resection of the thrombosed section of the artery, the patient fully recovered with no residual pain or limitations in the use of the affected arm. His patient-reported outcomes improved as well. It is important to investigate the existence of persistent median artery thrombosis if a patient presents with atypical symptoms of carpal tunnel syndrome. Ultrasonography is useful for the diagnosis of persistent median artery thrombosis. Surgical resection of a thrombosed persistent median artery in patients with carpal tunnel syndrome yields good results.

α-Lipoic acid eliminates dioxin-induced offspring sexual immaturity by improving abnormalities in folic acid metabolism.

Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes developmental and reproductive disorders in pups due to the attenuated luteinizing hormone (LH) production during the perinatal stage; however, the administration of α-lipoic acid (LA) to TCDD-exposed pregnant rats reversed the attenuated LH production. Therefore, reproductive disorders in pups are expected to be ameliorated with LA supplementation. To address this issue, pregnant rats orally received low dose TCDD at gestational day 15 (GD15) and proceeded to parturition. The control received a corn oil vehicle. To examine the preventive effects of LA, supplementation with LA was provided until postnatal day 21. In this study, we demonstrated that maternal administration of LA restored the sexually dimorphic behavior of male and female offspring. TCDD-induced LA insufficiency is likely a direct cause of TCDD reproductive toxicity. In the analysis to clarify the mechanism of the decrease in LA, we found evidence suggesting that TCDD inhibits the synthesis and increases the utilization of S-adenosylmethionine (SAM), a cofactor for LA synthesis, resulting in a decrease in the SAM level. Furthermore, folate metabolism, which is involved in SAM synthesis, is disrupted by TCDD, which may adversely affect infant growth. Maternal supplementation of LA restored SAM to its original level in the fetal hypothalamus; in turn, SAM ameliorated abnormal folate consumption and suppressed aryl hydrocarbon receptor activation induced by TCDD. The study demonstrates that the application of LA could prevent and recover next-generation dioxin reproductive toxicity, which provides the potential to establish effective protective measures against dioxin toxicity.

Improvement of Imaging Conditions to Improve the Detection Rate of Head and Neck Cancer by Positron Emission Tomography/Computed Tomography Examination.

Positron emission tomography (PET)/computed tomography (CT) has improved sensitivity and resolution using silicon photomultiplier as a photosensor. Previously, only a fixed setting was available for the shooting time of 1 bed, but now, the shooting time can be changed for each bed. Time can be shortened or extended depending on the target area. A few studies reported on image reconstruction conditions for head and neck cancer in whole-body PET/CT examinations. Thus, this study aimed to optimize the imaging conditions of the head and neck region during whole-body imaging. A cylindrical acrylic container with a 200 mm diameter was used to simulate the head and neck area using a PET/CT system equipped with a semiconductor detector. Spheres of 6-30 mm in diameter were enclosed in the 200 mm diameter cylindrical acrylic vessel. Radioactivity in 18F solution (Hot:BG ratio 4:1) was enclosed in a phantom following the Japanese Society of Nuclear Medicine (JSNM) guidelines. Background radioactivity concentration was 2.53 kBq/mL. List mode acquisition of 1,800 s was collected at 60-1,800 s with the field of view of 700 mm and 350 mm. The image was reconstructed by resizing the matrix to 128 × 128, 192 × 192, 256 × 256, and 384 × 384, respectively. The imaging time per bed in the head and neck should be at least 180 s, and the reconstruction conditions should be a field of view (FOV) of 350 mm, matrix sizes of ≥ 192, and a Bayesian penalized likelihood (BPL) reconstruction with a ß-value of 200. This allows detection of > 70% of the 8-mm spheres in the images.

Identification of genes responsible for absorbing palladium ion in Escherichia coli.

The capability of Escherichia coli BW25113 to adsorb palladium (Pd) ions in a single-gene-knockout library was investigated using high-throughput screening. The results revealed that compared to BW25113, nine strains promoted Pd ion adsorption, whereas 22 strains repressed. Although further studies are required because of the first screening results, our results will provide a new perspective for improving the biosorption.

Morphological evolution and diversity of pectoral fin skeletons in teleosts.

The Teleostei class has the most species of the fishes. Members of this group have pectoral fins, enabling refined movements in the water. Although teleosts live in a diverse set of environments, the skeletal pattern of pectoral fins in teleosts is considered to show little morphological variability. Here, in order to elucidate variations in pectoral fin skeletons and to identify their evolutionary processes, we compared the pectoral fin skeletons from 27 species of teleosts. We identified several variations and a diversity of pectoral fin skeletal patterns within some teleost groups. Taken together with previous reports on teleost skeletons, our findings reveal that in the course of teleost evolution, there are a mixture of conserved and non-conserved components in the pectoral fin skeletons of teleosts, and that teleosts may have experienced the variation and conservation of the number and shape of the proximal radials, the loss of the mesocoracoid, and the change in the distal radial-fin ray relationship.

Outcomes of Rerouting the Palmaris Longus to Extensor Pollicis Longus Tendon Transfer through the Second Extensor Compartment for Radial Nerve Palsy.

Background: A palmaris longus (PL) to extensor pollicis longus (EPL) is a standard tendon transfer used to restore thumb extension in patients with radial nerve palsy. This transfer is done by withdrawing the EPL from the third compartment and passing it subcutaneously to reach the PL. We modified this transfer by rerouting the EPL through the second extensor compartment to improve the retropulsion of the thumb. The aim of this study is to report the outcomes of this modified transfer. Methods: Four patients with traumatic radial nerve palsy underwent the modified PL to EPL transfer. They also underwent transfer of the pronator teres (PT) to extensor carpi radialis brevis (ECRB) and flexor carpi radialis (FCR) to extensor digitorum communis (EDC). Patients were followed up for at least 1 year after surgery. The data with regard to age, gender, cause of radial nerve palsy, duration between injury and surgery, and duration of follow-up was recorded. At final follow-up, the arc of motion at the interphalangeal joint (IPJ), metacarpophalangeal joint (MCPJ), palmar and radial abduction and retropulsion were measured for the reconstructed thumb and contralateral normal thumb. Results: All patients were male, with a mean age of 34.3 (range, 19-46) years. The mean duration between the injury and surgery was 15.9 (7-27) months, and the mean post-operative follow-up period was 16.8 (12-25) months. All patients recovered good thumb function. The mean arc of motion of the affected and contralateral thumb were IPJ flexion: 52°/80°; IPJ extension: 21°/14°; MCPJ flexion: 30°/33°; MCPJ extension:24°/31°; radial abduction: 70°/74°; palmar abduction: 68°/75° and retropulsion: 4.8cm/5.0cm. Conclusion: Rerouting the PL to EPL tendon transfer through the second extensor compartment in radial nerve palsy can restore good thumb function especially retropulsion. Level of Evidence: Level IV (Therapeutic).

Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4.

Heterologous expression systems are important for analyzing the effects of genetic factors including single nucleotide polymorphisms on the functions of drug-metabolizing enzymes. In this study, we focused on a baculovirus-mammalian cell (Bac-Mam) expression system as a safer and more efficient approach for this purpose. The baculovirus-insect cell expression system is widely utilized in large-scale protein expression. Baculovirus has been shown to also infect certain mammalian cells, although the virus only replicates in insect cells. With this knowledge, baculovirus is now being applied in a mammalian expression system called the Bac-Mam system wherein a gene-modified baculovirus is used whose promotor is replaced with one that can function in mammalian cells. We subcloned open-reading frames of cytochrome P450 3A4 (CYP3A4), UDP-glucuronosyltransferase (UGT) 1A1, and UGT2B7 into a transfer plasmid for the Bac-Mam system, and prepared recombinant Bac-Mam virus. The obtained virus was amplified in insect Sf9 cells and used to infect mammalian COS-1 cells. Expression of CYP3A4, UGT1A1, and UGT2B7 in COS-1 cell homogenates were confirmed by immunoblotting. Optimum infection conditions including the amount of Bac-Mam virus, culture days before collection, and concentration of sodium butyrate, an enhancer of viral-transduction were determined by monitoring CYP3A4 expression. Expressed CYP3A4 showed appropriate activity without supplying hemin/5-aminolevulinic acid or co-expressing with NADPH-cytochrome P450 reductase. Further, we compared gene transfer efficiency between the Bac-Mam system and an established method using recombinant plasmid and transfection reagent. Our results indicate that the Bac-Mam system can be applied to introduce drug-metabolizing enzyme genes into mammalian cells that are widely used in drug metabolism research. The expressed enzymes are expected to undergo appropriate post-translational modification as they are in mammalian bodies. The Bac-Mam system may thus accelerate pharmacogenetics and pharmacogenomics research.

[Pulmonary Partial Resection for a Lung Foreign Body( Watermelon Seed):Report of a Case].

We experienced a case of a foreign body in the lung with granuloma by aspiration of watermelon seeds. A 72-year-old woman who had been diagnosed as having lung foreign body was admitted to our hospital for the treatment of the pulmonary shadow caused by the granuloma. A foreign body could not be identified by bronchoscopy, and the thoracoscopic partial resection of right S4 was performed. The postoperative course was uneventful, and the patient was discharged from our hospital on the second day after the operation.

Adenine-related compounds modulate UDP-glucuronosyltransferase (UGT) activity in mouse liver microsomes.

Adenine-related compounds are allosteric inhibitors of UDP-glucuronosyltransferase (UGT) in rat liver microsomes (RLM) and human UGT isoforms treated with detergent or pore-forming peptide, alamethicin.To clarify whether the same is true beyond species, the effects of adenine-related compounds on 4-methylumbelliferone (4-MU) glucuronidation were examined using detergent-treated mouse liver microsomes (MLM).Brij-58 treatment of MLM increased the Vmax and the Michaelis constant, Km, of 4-MU. This study was performed using Brij-58-treated MLM as an enzyme source. ATP- and ADP-inhibited 4-MU glucuronidation. In contrast, AMP caused a 1.5-fold increase in glucuronidation. Oxidised forms, NAD+ and NADP+, potently inhibited 4-MU glucuronidation, whereas the reduced forms, NADH and NADPH, did not. Furthermore, the IC50 values of ATP, ADP, NAD+, and NADP+ were approximately 15 µM.In our previous study, ATP was the strongest inhibitor of UGT activity in RLM. However, in this study, the above-mentioned compounds inhibited 4-MU UGT in a comparable and non-competitive manner. Furthermore, AMP antagonised the inhibitory effects of ATP and ADP.These results suggest that ATP, ADP, NAD+, and NADP+ are common endogenous inhibitors of UGT beyond species.

DAPL1 is a novel regulator of testosterone production in Leydig cells of mouse testis.

Leydig cells in the testes produce testosterone in the presence of gonadotropins. Therefore, male testosterone levels must oscillate within a healthy spectrum, given that elevated testosterone levels augment the risk of cardiovascular disorders. We observed that the expression of death-associated protein-like 1 (DAPL1), which is involved in the early stages of epithelial differentiation and apoptosis, is considerably higher in the testes of sexually mature mice than in other tissues. Accordingly, Dapl1-null mice were constructed to evaluate this variation. Notably, in these mice, the testicular levels of steroidogenic acute regulatory protein (StAR) and serum testosterone levels were significantly elevated on postnatal day 49. The findings were confirmed in vitro using I-10 mouse testis-derived tumor cells. The in vivo and in vitro data revealed the DAPL1-regulated the expression of StAR involving altered transcription of critical proteins in the protein kinase A and CREB/CREM pathways in Leydig cells. The collective findings implicate DAPL1 as an important factor for steroidogenesis regulation, and DAPL1 deregulation may be related to high endogenous levels of testosterone.

Ablation of Selenbp1 Alters Lipid Metabolism via the Pparα Pathway in Mouse Kidney.

Selenium-binding protein 1 (Selenbp1) is a 2,3,7,8-tetrechlorodibenzo-p-dioxin inducible protein whose function is yet to be comprehensively elucidated. As the highly homologous isoform, Selenbp2, is expressed at low levels in the kidney, it is worthwhile comparing wild-type C57BL mice and Selenbp1-deficient mice under dioxin-free conditions. Accordingly, we conducted a mouse metabolomics analysis under non-dioxin-treated conditions. DNA microarray analysis was performed based on observed changes in lipid metabolism-related factors. The results showed fluctuations in the expression of numerous genes. Real-time RT-PCR confirmed the decreased expression levels of the cytochrome P450 4a (Cyp4a) subfamily, known to be involved in fatty acid ω- and ω-1 hydroxylation. Furthermore, peroxisome proliferator-activated receptor-α (Pparα) and retinoid-X-receptor-α (Rxrα), which form a heterodimer with Pparα to promote gene expression, were simultaneously reduced. This indicated that reduced Cyp4a expression was mediated via decreased Pparα and Rxrα. In line with this finding, increased levels of leukotrienes and prostaglandins were detected. Conversely, decreased hydrogen peroxide levels and reduced superoxide dismutase (SOD) activity supported the suppression of the renal expression of Sod1 and Sod2 in Selenbp1-deficient mice. Therefore, we infer that ablation of Selenbp1 elicits oxidative stress caused by increased levels of superoxide anions, which alters lipid metabolism via the Pparα pathway.

LAPTM4α is targeted from the Golgi to late endosomes/lysosomes in a manner dependent on the E3 ubiquitin ligase Nedd4-1 and ESCRT proteins.

Lysosome-associated protein transmembrane 4α (LAPTM4α) is a four transmembrane-spanning protein primarily localized in endosomes and lysosomes and has several putative lysosomal targeting signals at its C-terminal cytoplasmic domain, including tyrosine-based motifs (YxxΦ) and PY motifs (L/PxxY). LAPTM4α has been previously shown to be ubiquitinated by the E3 ubiquitin ligase Nedd4-1 through binding to its PY motifs and sorted to lysosomes, however, the molecular mechanisms underlying the localization of LAPTM4α to endosomes/lysosomes have not yet been fully elucidated. In the present study, we show that LAPTM4α binds Nedd4-1 in a manner dependent on PY motifs, while the PY motifs and Nedd4-1 are not necessarily required for LAPTM4α ubiquitination. The binding of LAPTM4α with Nedd4-1, however, is necessary for an effective sorting of LAPTM4α from the Golgi to late endosomes/lysosomes. An unexpected finding is that LAPTM4α is localized in the lumen, but not in the limiting membrane, of late endosomes, and degraded in lysosomes over time. Interestingly, we further found that siRNA knockdown of endosomal sorting complexes required for transport (ESCRT) components that mediate sorting of ubiquitinated membrane proteins into intralumenal vesicles (ILVs) of endosomes selectively blocks the transport of LAPTM4α to endosomes. Collectively, these results suggest that trafficking of LAPTM4α from the Golgi to endosomes is promoted by the interaction with Nedd4-1, which further requires ESCRT components. Furthermore, our findings highlight a novel function for ESCRT proteins in mediating protein and/or vesicle trafficking from the Golgi to endosomes/lysosomes.

An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Evaluation of dose reduction potential in scatter-corrected bedside chest radiography using U-net.

Bedside radiography has increasingly attracted attention because it allows for immediate image diagnosis after X-ray imaging. Currently, wireless flat-panel detectors (FPDs) are used for digital radiography. However, adjustment of the X-ray tube and FPD alignment are extremely difficult tasks. Furthermore, to prevent a poor image quality caused by scattered X-rays, scatter removal grids are commonly used. In this study, we proposed a scatter-correction processing method to reduce the radiation dose when compared with that required by the X-ray grid for the segmentation of a mass region using deep learning during bedside chest radiography. A chest phantom and an acrylic cylinder simulating the mass were utilized to verify the image quality of the scatter-corrected chest X-rays with a low radiation dose. In addition, we used the peak signal-to-noise ratio and structural similarity to quantitatively assess the quality of the low radiation dose images compared with normal grid images. Furthermore, U-net was used to segment the mass region during the scatter-corrected chest X-ray with a low radiation dose. Our results showed that when scatter correction is used, an image with a quality equivalent to that obtained by grid radiography is produced, even when the imaging dose is reduced by approximately 20%. In addition, image contrast was improved using scatter radiation correction as opposed to using scatter removal grids. Our results can be utilized to further develop bedside chest radiography systems with reduced radiation doses.

The carboxyl-terminal di-lysine motif is essential for catalytic activity of UDP-glucuronosyltransferase 1A9.

UDP-Glucuronosyltransferase (UGT) is a type I membrane protein localized to the endoplasmic reticulum (ER). UGT has a di-lysine motif (KKXX/KXKXX) in its cytoplasmic domain, which is defined as an ER retention signal. However, our previous study has revealed that UGT2B7, one of the major UGT isoform in human, localizes to the ER in a manner that is independent of this motif. In this study, we focused on another UGT isoform, UGT1A9, and investigated the role of the di-lysine motif in its ER localization, glucuronidation activity, and homo-oligomer formation. Immunofluorescence microscopy indicated that the cytoplasmic domain of UGT1A9 functioned as an ER retention signal in a chimeric protein with CD4, but UGT1A9 itself could localize to the ER in a di-lysine motif-independent manner. In addition, UGT1A9 formed homo-oligomers in the absence of the motif. However, deletion of the di-lysine motif or substitution of lysines in the motif for alanines, severely impaired glucuronidation activity of UGT1A9. This is the first study that re-defines the cytoplasmic di-lysine motif of UGT as an essential peptide for retaining glucuronidation capacity.

HM1.24/BST-2 is constitutively poly-ubiquitinated at the N-terminal amino acid in the cytoplasmic domain.

HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery.