Pimonidazole-alkyne conjugate for sensitive detection of hypoxia by Cu-catalyzed click reaction.

Hypoxia is involved in various diseases, such as cancers. Pimonidazole has often been used as the gold-standard marker to visualize hypoxic regions. Pimonidazole labels hypoxic regions by forming a covalent bond with a neighboring protein under hypoxic conditions in the body, which is detected by immunohistochemistry performed on tissue sections. To date, some pimonidazole-fluorophore conjugates have been reported as fluorescent probes for hypoxia imaging that do not require immunostaining. They are superior to pimonidazole because immunostaining can produce high background signals. However, large fluorophores in the conjugates may alter the original biodistribution and reactivity. Here, we report a new hypoxia marker, Pimo-yne, as a pimonidazole-alkyne conjugate. Pimo-yne has a similar hypoxia detection capability as pimonidazole because the alkyne tag is small and can be detected by Cu-catalyzed click reaction with azide-tagged fluorescent dyes. We successfully visualized hypoxic regions in tumor tissue sections using Pimo-yne with reduced background signals. The detected regions overlapped well with those detected by pimonidazole immunohistochemistry. To further reduce the background, we employed a turn-on azide-tagged fluorescent dye.

Whole-Body and Whole-Organ 3D Imaging of Hypoxia Using an Activatable Covalent Fluorescent Probe Compatible with Tissue Clearing.

Elucidation of biological phenomena requires imaging of microenvironments in vivo. Although the seamless visualization of in vivo hypoxia from the level of whole-body to single-cell has great potential to discover unknown phenomena in biological and medical fields, no methodology for achieving it has been established thus far. Here, we report the whole-body and whole-organ imaging of hypoxia, an important microenvironment, at single-cell resolution using activatable covalent fluorescent probes compatible with tissue clearing. We initially focused on overcoming the incompatibility of fluorescent dyes and refractive index matching solutions (RIMSs), which has greatly hindered the development of fluorescent molecular probes in the field of tissue clearing. The fluorescent dyes compatible with RIMS were then incorporated into the development of activatable covalent fluorescent probes for hypoxia. We combined the probes with tissue clearing, achieving comprehensive single-cell-resolution imaging of hypoxia in a whole mouse body and whole organs.

Hypoxia activates SREBP2 through Golgi disassembly in bone marrow-derived monocytes for enhanced tumor growth.

Bone marrow-derived cells (BMDCs) infiltrate hypoxic tumors at a pre-angiogenic state and differentiate into mature macrophages, thereby inducing pro-tumorigenic immunity. A critical factor regulating this differentiation is activation of SREBP2-a well-known transcription factor participating in tumorigenesis progression-through unknown cellular mechanisms. Here, we show that hypoxia-induced Golgi disassembly and Golgi-ER fusion in monocytic myeloid cells result in nuclear translocation and activation of SREBP2 in a SCAP-independent manner. Notably, hypoxia-induced SREBP2 activation was only observed in an immature lineage of bone marrow-derived cells. Single-cell RNA-seq analysis revealed that SREBP2-mediated cholesterol biosynthesis was upregulated in HSCs and monocytes but not in macrophages in the hypoxic bone marrow niche. Moreover, inhibition of cholesterol biosynthesis impaired tumor growth through suppression of pro-tumorigenic immunity and angiogenesis. Thus, our findings indicate that Golgi-ER fusion regulates SREBP2-mediated metabolic alteration in lineage-specific BMDCs under hypoxia for tumor progression.

Radiographic Evaluation after Arthroscopic Partial Trapeziectomy with Suture-button Suspensionplasty for Thumb Carpometacarpal Arthritis.

Arthroscopic partial trapeziectomy with suture-button suspensionplasty was developed for the surgical treatment of thumb carpometacarpal arthritis. However, the relationship between clinical results and radiographic evidence is unclear. Methods: The authors retrospectively reviewed 33 consecutive patients who underwent arthroscopic partial trapeziectomy with suture-button suspensionplasty for thumb carpometacarpal arthritis between 2016 and 2021. Clinical and radiographic outcomes were recorded, and the correlations between them were evaluated. Results: The average patient age at surgery was 69 years. Patient radiologic evidence was Eaton stage Ⅱ in three thumbs, Ⅲ in 25 thumbs, and Ⅳ in five thumbs. The average trapezial space ratio (TSR) was 0.36 immediately after the operation but declined to 0.32 after 6 months. In contrast, the average joint subluxation was reduced to 0.005 immediately after the operation compared with 0.28 before, and was maintained at 0.04 at final follow-up. A statically significant correlation was detected between grip strength and TSR (P = 0.03), and between pinch strength and TSR (P = 0.02). A significant correlation was detected between TSR and trapezium height (P = 0.0215), which remained after partial trapeziectomy. No correlation was detected between rope position and other clinical or radiographic scores. Conclusions: Suture-button can have an effect on the medialization of the first metacarpal base. Excessive trapeziectomy can result in functional deficiency of the thumb through metacarpal subsidence, which potentially causes loss of grip and pinch strength.

Clinical Results of Arthroscopic Partial Trapeziectomy With Suture-Button Suspensionplasty for Thumb Carpometacarpal Arthritis.

BACKGROUND: This study represents the clinical results, especially range of motion (ROM) improvement, of arthroscopic partial trapeziectomy with suture-button suspensionplasty for symptomatic grade II and III thumb carpometacarpal arthritis with a minimum 1-year follow-up. METHODS: Thirty-two patients (mean: 67.5 years) with grade II and III thumb carpometacarpal arthritis treated with arthroscopic partial trapeziectomy with suture-button suspensionplasty were retrospectively followed up for at least 1 year. The physical assessments included ROM, pain visual analogue scale (VAS), strength, and the Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire. The physical variables were retrospectively compared before surgery and at the final follow-up. RESULTS: Preoperative radial abduction and palmar abduction (45.4 ± 16.4° and 54.3 ± 13.9°, respectively) were significantly increased at the final follow-up (59.7 ± 16.9° and 65.5 ± 14.2°, respectively). Preoperative VAS score, pinch strength, and DASH score (70.5 ± 14.0, 57.2 ± 24.8% and 36.8 ± 14.8, respectively) were also significantly improved at the final follow-up (7.9 ± 9.1, 91.0 ± 39.6%, and 11.7 ± 10.5, respectively). Complications involved 1 case of irritation of the superficial branch of the radial nerve and 1 case of dystonia. Two suture-buttons were removed due to patient discomfort. CONCLUSIONS: A significant increase in ROM and pain relief was obtained after suture-button suspensionplasty with arthroscopic partial trapeziectomy.

Muscle Grafts with Doxorubicin Pretreatment Produce "Empty Tubes" in the Basal Laminae, Promote Contentious Maturation of the Regenerated Axons, and Bridge 20-mm Sciatic Nerve Defects in Rats.

BACKGROUND: We newly developed a muscle graft that employs a doxorubicin pretreatment technique. The aims of this study were to reveal the biological and morphological features of the muscle tissue in the second week (Study I), to reveal the regeneration outcomes of functional and kinematic assessments of longer-term follow-up (16 weeks, Study II), and to make assessments of the muscle graft with doxorubicin pretreatment in the critical-sized nerve defect model (20 mm, Study III). METHODS: A total of 26 adult rats were used in this study. Doxorubicin treatment was accomplished by immersion in a doxorubicin solution for 10 minutes followed by a rinsing procedure. The rats were divided into three groups: the muscle graft with and without doxorubicin pretreatment (M-graft-w-Dox and M-graft-w/o-Dox) groups and the autologous nerve graft (N-graft) group. Assays of apoptosis, immunofluorescent histochemistry including CD68 (macrophage marker), scanning electron microscopy (SEM), morphometrical studies of the regenerated axons, nerve conduction studies, and kinematic studies were performed. RESULTS: The M-graft-w-Dox group contained significantly larger numbers of apoptotic cells and CD68-positive cells. SEM revealed the existence of the basal lamina, so called "empty tubes," in the M-graft-w-Dox group. Study II showed contentious maturation of the regenerated axons, especially in the compound muscle action potentials. Study III showed that even at 20 mm, the M-graft-w-Dox group promoted axonal regeneration and functional regeneration. CONCLUSION: The M-graft-w-Dox group showed superior regeneration results, and this easy and short-term procedure can expand the muscle graft clinical indication for the treatment of peripheral nerve defects.

Anterolateral thigh flap for upper extremity reconstruction in older patients.

BACKGROUND: The anterolateral thigh (ALT) flap has been used in upper extremity reconstruction. However, there is no consensus about the age at which the flap can be used safely, which is a concern when applying ALT flaps for upper extremity reconstruction in older patients. We present the results of the use of ALT flap for upper extremity reconstruction in a series of older patients. PATIENTS AND METHODS: Seventeen patients who underwent ALT flaps for soft tissue defects in the upper extremities from 2010 to 2020 were included. The patients' mean age was 63.5 (range, 26-83) years. Ten of seventeen patients were smokers. Defect locations were the dorsum of the hand in seven patients, palm in two patients, dorsum and palm in two patients, and forearm in six patients. Etiologies of the defect were traumatic in 14 patients and malignant tumor in three patients. The defect size was 8 to 25 × 5 to 11 cm. When dissecting the perforators, we preserved the surrounding small muscular and fatty tissue with the perforators and to harvest them together to prevent intima damage. Flap thinning was performed for 16 flaps to adjust the flap thickness to match defect site requirements. We used an end-to-side or interposition arterial anastomosis to regulate the blood flow. RESULTS: The flap size was 9 to 28 × 5 to 13 cm. One patient had venous congestion and vein re-anastomosis was needed. All flaps survived. One patient had a methicillin-resistant Staphylococcus aureus infection and debridement and irrigation was needed. The mean follow-up period was 20 (range, 13-37) months. Fifteen patients returned their previous activities. The mean DASH score was 30.6 (range, 3-70). CONCLUSIONS: Regardless of patient age or smoking status, the ALT flap was a safe and reliable surgical option for soft tissue defect reconstruction of the upper extremity.

Nerve regeneration using the Bio 3D nerve conduit fabricated with spheroids.

Autologous nerve grafting is the gold standard method for peripheral nerve injury with defects. Artificial nerve conduits have been developed to prevent morbidity at the harvest site. However, the artificial conduit regeneration capacity is not sufficient. A Bio 3D printer is technology that creates three-dimensional tissue using only cells. Using this technology, a three-dimensional nerve conduit (Bio 3D nerve conduit) was created from several cell spheroids. We reported the first application of the Bio 3D nerve conduit for peripheral nerve injury. A Bio 3D nerve conduit that was created from several cells promotes peripheral nerve regeneration. The Bio 3D nerve conduit may be useful clinically to treat peripheral nerve defects.

Enthesopathy of the bicipital tuberosity of the radius treated under intraoperative computed tomography.

BACKGROUND: There is no report of the application of intraoperative computed tomography to the extremities, and its usefulness is not mentioned. CASE PRESENTATION: We present a case of a patient with the elbow pain and loss of the forearm rotation due to the prominent bicipital tuberosity of the radius, which was diagnosed as enthesopathy. Surgical treatment to excise the prominent part of the bicipital tuberosity of the radius was recommended. However, it is difficult to perform the appropriate excision of the abnormal prominent part because of complications such as bicipital tendon rupture. The patient was successfully treated by surgical resection under the control of intraoperative computed tomography. CONCLUSIONS: Intraoperative computed tomography scan is a useful tool to assess the remaining volume of the abnormal bones.

The efficacy of combining a vascularized biogenic conduit and a decellularized nerve graft in the treatment of peripheral nerve defects: An experimental study using the rat sciatic nerve defect model.

BACKGROUND: Although decellularized nerve grafts are often used as a bridging material in nerve defect repair, the lack of perfusion support in this procedure limits the regeneration capacity. To address this, we applied vascularized biogenic conduits, which are fibrous membranes prefabricated around the silicone rod that contain rich vascularity and macrophages, to nerve defect repair procedures using decellularized nerve grafts. The purpose of this study is to investigate the capacity of combining a vascularized biogenic conduit and a decellularized nerve graft for peripheral nerve regeneration using a 10-mm nerve defect model in rats. MATERIALS AND METHODS: Sixteen adult male rats (F344 rats, 10-12 weeks, 200-250 g) were used in this study. For the prefabrication of vascularized biogenic conduits, a silicone rod was transplanted next to the sciatic nerve. After 8 weeks, this silicone rod was enveloped in connective tissue, called a vascularized biogenic conduit. The first rat was used to investigate the histological characteristics of vascularized biogenic conduits through immunofluorescence studies. The remaining 15 rats were divided into three groups to evaluate the efficacy of the combination of a decellularized nerve graft and a vascularized biogenic conduit: a decellularized nerve graft (DNG) group, a decellularized nerve graft with a vascularized biogenic conduit (DNG-w-VBC) group, and an autologous nerve graft (ANG) group. Eight weeks after nerve graft surgery, the assessment results of both functional recovery (electrophysical studies and target muscle atrophy) and morphological recovery (total number, diameter, and myelin thickness of the regenerated axons) of the regenerated nerves were examined. RESULTS: Immunofluorescence studies revealed that the VBC contains extracellular matrix, vascular tissue, and macrophages. The results of the DNG-w-VBC group were superior to the DNG group in electrophysiological studies (CMAP; 6.29 ± 0.80% vs. 4.02 ± 3.35%, MNCV; 50.6 ± 8.4% vs. 25.7 ± 15.6%, p < .05, respectively), regenerated axon number (11,348 ± 812 vs. 7697 ± 2197, p < .05), and mean axon diameter (2.72 ± 0.33 µm vs. 1.64 ± 0.12 µm, p < .05). CONCLUSIONS: Our study confirms that vascularized biogenic conduits supply vascularity and macrophages to nerve defect sites. Combining vascularized biogenic conduits with decellularized nerve grafts to treat nerve defects offers superior functional and morphological recovery of regenerated axons.

The apicoplast genome of Leucocytozoon caulleryi, a pathogenic apicomplexan parasite of the chicken.

Leucocytozoon caulleryi, a haemosporidian parasite of the chicken (Gallus gallus domesticus), can be highly pathogenic and often fatal. Although this parasite is extremely relevant to veterinary science, knowledge of its genomic features is limited. To gain information applicable to developing novel control methods for the parasite, we analyzed the apicoplast genome of L. caulleryi. This extranuclear organellar DNA of 85.1% A + T and a unit of 34,779 bp was found to encode almost the same set of genes as the plastid genome of Plasmodium falciparum, including 16 tRNA and 30 protein coding genes, and except for one open reading frame, ORF91 absent in L. caulleryi. As in P. falciparum, the L. caulleryi apicoplast DNA contains two sets of a unique inverted repeat (IR), each one 5,253 bp and encoding genes specifying one large and one small rRNA subunit and nine tRNAs but no protein, and separated by a unique 13 bp sequence. Studies of several haemosporidian apicoplast DNA sequences have identified a corresponding IR region; however, none of these studies has looked at the complete sequence, even for well-studied species such as P. falciparum. Phylogenetic studies using a concatenated amino acid sequence based on the open reading frames confirmed the close relationship between L. caulleryi and Plasmodium spp. In this study, we determined the nucleotide sequence of the entire L. caulleryi apicoplast genome, including the region connecting the two IR units. This is the first report of the complete nucleotide sequence of a haemosporidian apicoplast DNA with a canonical IR.