Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dß1, and Dß2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dß3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dß3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Extrahepatic approach for taping the common trunk of the middle and left hepatic veins or the left hepatic vein alone in laparoscopic hepatectomy (with videos).

BACKGROUND: Outflow control is difficult, and techniques required for effectively handling intraoperative hemorrhage during laparoscopic hepatectomy have not previously been adequately reported. METHODS: Sixteen patients underwent surgery, of which 15 underwent laparoscopic left hepatectomy and one underwent laparoscopic partial hepatectomy of the caudate lobe. Encircling and taping of the common trunk of the middle (MHV) and left hepatic veins (LHV) was performed in 12 patients, and that of the LHV alone in four patients. Surgical techniques based on anatomical landmarks and histological findings are presented with videos. Histological confirmation of the anatomical landmarks for these procedures was performed in fresh cadavers to understand the anatomical structures and layers involved. RESULTS: The median procedure duration was 15 (6-25) minutes. All procedures were performed safely with no major bleeding. Histological findings showed fibrous connective tissue between the tunica adventitia of the inferior vena cava (IVC) and the Laennec's capsule of the liver. The layer of dissection was along the tunica adventitia of the IVC. CONCLUSIONS: The surgical techniques for encircling and taping of the common trunk of the MHV and LHV and the LHV alone based on anatomical landmarks were feasible and could allow for efficient outflow control in laparoscopic hepatectomy.

Admp regulates tail bending by controlling ventral epidermal cell polarity via phosphorylated myosin localization in Ciona.

Ventral tail bending, which is transient but pronounced, is found in many chordate embryos and constitutes an interesting model of how tissue interactions control embryo shape. Here, we identify one key upstream regulator of ventral tail bending in embryos of the ascidian Ciona. We show that during the early tailbud stages, ventral epidermal cells exhibit a boat-shaped morphology (boat cell) with a narrow apical surface where phosphorylated myosin light chain (pMLC) accumulates. We further show that interfering with the function of the BMP ligand Admp led to pMLC localizing to the basal instead of the apical side of ventral epidermal cells and a reduced number of boat cells. Finally, we show that cutting ventral epidermal midline cells at their apex using an ultraviolet laser relaxed ventral tail bending. Based on these results, we propose a previously unreported function for Admp in localizing pMLC to the apical side of ventral epidermal cells, which causes the tail to bend ventrally by resisting antero-posterior notochord extension at the ventral side of the tail.

Spatiotemporal dynamics of single cell stiffness in the early developing ascidian chordate embryo.

During the developmental processes of embryos, cells undergo massive deformation and division that are regulated by mechanical cues. However, little is known about how embryonic cells change their mechanical properties during different cleavage stages. Here, using atomic force microscopy, we investigated the stiffness of cells in ascidian embryos from the fertilised egg to the stage before gastrulation. In both animal and vegetal hemispheres, we observed a Rho kinase (ROCK)-independent cell stiffening that the cell stiffness exhibited a remarkable increase at the timing of cell division where cortical actin filaments were organized. Furthermore, in the vegetal hemisphere, we observed another mechanical behaviour, i.e., a ROCK-associated cell stiffening, which was retained even after cell division or occurred without division and propagated sequentially toward adjacent cells, displaying a characteristic cell-to-cell mechanical variation. The results indicate that the mechanical properties of embryonic cells are regulated at the single cell level in different germ layers.

Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

Different strategies for tissue scaling in dwarf tailbud embryos revealed by single-cell analysis.

The tailbud stage is part of the organogenesis period-an evolutionarily conserved developmental period among chordates that is essential for determining the characteristics of the chordate body plan. When the volume of the egg is artificially decreased by cutting, ascidians produce a normal-looking but miniature (dwarf) tailbud embryo. Although cell lineages during ascidian embryogenesis are invariant, the number of cell divisions in the dwarf embryo is altered by a different mechanism in each tissue (Yamada and Nishida, 1999). Here, to elucidate the size-regulation strategies of the Ciona robusta dwarf tailbud embryo, we compared anatomical structure, developmental speed, and cell number/volume in each tissue between dwarf and wild type (WT) embryos. To do this, we constructed a 3D virtual mid-tailbud embryo (Nakamura et al., 2012). We could make a Ciona dwarf tailbud embryo from eggs with a diameter over 108 â€‹µm (correspond to â€‹> â€‹40% of the wild type egg volume). The timings of cleavage (~St. 12) and subsequent morphogenesis were nearly the same but blastomeres of animal hemisphere slightly delayed the timing of mitosis in the early cleavage period. Intriguingly, the tissue-to-tissue volume ratios of dwarf tailbud embryos were similar to those of wild type embryos suggesting that the ratio of tissue volumes is essential for maintaining the proper shape of the tailbud embryo. The number of cells in the epidermis, nervous system, and mesenchyme was significantly reduced in the dwarf embryos whereas the cell volume distribution of these tissues was similar in the dwarf and wild type. In contrast, the number of cells in the notochord, muscle, heart, and endoderm were maintained in the dwarf embryos; cell volumes were significantly reduced. Neither parameter changed in germline precursors. These results indicate that each tissue uses different scaling strategies to coordinate cell number and cell volume in accordance with the embryo size.

Successful resection of pancreatic metastasis from oesophageal squamous cell carcinoma: a case report and review of the literature.

BACKGROUND: Oesophageal cancer has a high metastatic potential and poor prognosis, with a significant risk of recurrence after radical resection. However, resected pancreatic metastasis from oesophageal cancer is rare. CASE PRESENTATION: Eleven years prior, a seventy-year-old woman had been treated with transthoracic radical oesophagectomy for oesophageal squamous cell carcinoma. Four years prior, she had undergone chemotherapy for lymph node recurrence at the splenic hilum and achieved a partial response. She had also received chemoradiotherapy for lymph node recurrence at the splenic hilum 3 years prior; a complete response was achieved. However, routine follow-up with abdominal computed tomography recently revealed a tumour at the pancreatic tail and swollen lymph nodes. The patient underwent distal pancreatectomy on the basis of a pre-operative diagnosis of primary pancreatic cancer, although a histological examination of the surgical specimen revealed metastatic squamous cell carcinoma that was compatible with metachronous pancreatic metastasis from oesophageal squamous cell carcinoma. The patient has been undergoing clinical follow-up without adjuvant therapy and has been disease-free for 24 months after resection of the pancreatic metastasis. CONCLUSIONS: Resection of pancreatic metastasis may improve prognosis and should be considered when treating patients with solitary metastasis from oesophageal squamous cell carcinoma.

Two-stage resection of a bilateral pheochromocytoma and pancreatic neuroendocrine tumor in a patient with von Hippel-Lindau disease: A case report.

INTRODUCTION: von Hippel-Lindau disease (vHL disease) is a hereditary disease in which tumors and cysts develop in many organs, in association with central nervous system hemangioblastomas, pheochromocytomas, and pancreatic tumors. We herein report a case of vHL disease (type 2A) associated with bilateral pheochromocytomas, pancreatic neuroendocrine tumors (PNET), and cerebellar hemangioblastomas treated via pancreatectomy after adrenalectomy. CASE PRESENTATION: A 51-year-old woman presented with a cerebellar tumor, bilateral hypernephroma, and pancreatic tumor detected during a medical checkup. 18F-fluorodeoxyglucose positron emission tomography-computed tomography revealed a bilateral adrenal gland tumor and a tumor in the head of the pancreas, while an abdominal computed tomography examination revealed a 30-mm tumor with strong enhancement in the head of the pancreas. Cranial magnetic resonance imaging showed a hemangioblastoma in the cerebellum. Therefore, a diagnosis of vHL disease (type 2A) was made. Her family medical history included renal cell carcinoma in her father and bilateral adrenal pheochromocytoma and spinal hemangioblastoma in her brother. A detailed examination of endocrine function showed that the adrenal mass was capable of producing catecholamine. Treatment of the pheochromocytoma was prioritized, and therefore, laparoscopic left adrenalectomy and subtotal resection of the right adrenal gland were performed. Once the postoperative steroid levels were replenished, subtotal stomach-preserving pancreatoduodenectomy was performed for the PNET. After a good postoperative course, the patient was discharged in remission on the 11th day following surgery. Histopathological examination findings indicated NET G2 (MIB-1 index 10-15%) pT3N0M0 Stage II A and microcystic serous cystadenoma throughout the resected specimen. The patient is scheduled to undergo treatment for the cerebellar hemangioblastoma. CONCLUSION: A two-staged resection is a safe and effective treatment option for bilateral pheochromocytoma and PNET associated with vHL disease.

Tip-Apex Distance Is Most Important of Six Predictors of Screw Cutout After Internal Fixation of Intertrochanteric Fractures in Women.

BACKGROUND: Six risk factors for screw cutout after internal fixation of intertrochanteric fractures have been reported. The purpose of the present study was to evaluate and compare the impact of the 6 risk factors of screw cutout to clarify the most important one. METHODS: We enrolled 8 consecutive patients who had screw cutout and 48 random control subjects after internal fixation of intertrochanteric fractures treated with proximal femoral nail antirotation systems at our institution. All of the patients were female. The group that had screw cutout and the control group were retrospectively evaluated and compared with respect to the OTA/AO classification, presence of a posterolateral fragment, types of reduction pattern on anteroposterior and lateral radiographic images, position of the screw, and the presence of a tip-apex distance (TAD) of ≥20 mm. The impact of each factor on screw cutout was assessed using backward stepwise multivariable logistic regression analysis with the Akaike information criterion. Risk stratification was assessed using classification and regression tree (CART) analysis. RESULTS: Among 6 risk factors, only a TAD of ≥20 mm had a significant impact on screw cutout, with an adjusted odds ratio of 12.4 (95% confidence interval, 1.6 to 129.0; p = 0.019). CART analysis also demonstrated that a TAD of ≥20 mm was the most important risk stratification factor (p < 0.001). CONCLUSIONS: Among the 6 previously reported screw cutout-related factors, only a TAD of ≥20 mm was associated with screw cutout after internal fixation of intertrochanteric fractures with proximal femoral nail antirotation systems. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

[Complete Response in Far-Advanced Esophageal Cancer Treated with Induction Chemotherapy Followed by Definitive Chemoradiotherapy].

We report a case of far-advanced esophageal cancer in which induction chemotherapy followed by chemoradiotherapy achieved complete remission. A 61-year-old female presented to our hospital with dyspnea and hoarseness. CT revealed a tumor at the cervical esophagus invading and narrowing the trachea, a bulky metastasis at the right paraesophageal node, and nodal metastases at levels II and III of the left neck. No finding indicated other distant metastases. According to findings of CT and endoscopy, she was diagnosed with unresectable cancer at the cervical esophagus(cT4bN1M1[LYM], according to UICC-TNM 7 th). After 2 courses of induction chemotherapy(DTX, CDDP, and 5-FU), the tumor's volume was remarkably reduced. Thereafter, chemoradiotherapy with CDDP, 5-FU, and 60 Gy/30 Fr was administered. After 7 months of systemic chemotherapy with paclitaxel following chemoradiotherapy, the patient was judged to have complete remission based on CT and endoscopic findings. After additional administration of S-1 for 5 months, systemic chemotherapy was ceased. The patient has survived without disease progression for 22 months following initiation of treatment. It is thought that induction chemotherapy followed by chemoradiotherapy might improve local control and survival of patients with far-advanced esophageal cancers, such as our patient.

Efficacy of preoperative skin traction for hip fractures: a single-institution prospective randomized controlled trial of skin traction versus no traction.

BACKGROUND: Preoperative traction for hip fractures is of no benefit in semi-urgent surgery. However, its efficacy has not been assessed in cases in which emergency surgery was not possible. We evaluated the efficacy of preoperative skin traction for hip fractures in a level II trauma center in Japan where many patients undergo delayed surgery. METHODS: We undertook a randomized controlled trial. Eighty-one patients were randomized to be treated with skin traction (41 patients), or bed rest (40 patients). Preoperative pain was assessed by use of a visual analogue scale and the number of analgesics required. Fracture reduction was measured on the basis of leg-length and neck-shaft angle discrepancies on the radiograph on admission, a day before surgery, and after surgery. RESULTS: The mean time from admission to surgery was 7.5 days. Pain decreased markedly on the day after admission in both the traction and no-traction groups. No significant difference was found during the preoperative waiting period between the groups in either pain score or number of analgesics taken. No significant difference was found in radiographic data either before or after surgery, and satisfactory reduction was achieved after surgery irrespective of the use of skin traction. CONCLUSIONS: In our single-institution prospective randomized controlled trial, preoperative skin traction for patients with hip fracture had no effect on pain relief before surgery or reduction of fracture displacement during surgery, irrespective of preoperative waiting time.