Potential therapeutic target secretogranin II might cooperate with hypoxia-inducible factor 1α in sunitinib-resistant renal cell carcinoma.

Multitargeted receptor tyrosine kinase inhibitors, including vascular endothelial growth factor (VEGF) inhibitors, such as sunitinib, have been used as the primary targeted agents for patients with recurrent or distant metastasis of advanced renal cell carcinoma (RCC). However, endogenous or acquired sunitinib resistance has become a significant therapeutic problem. Therefore, we focused on mechanisms of sunitinib resistance in RCC. First, we undertook RNA sequencing analysis using previously established sunitinib-resistant RCC (SUR-Caki1, SUR-ACHN, and SUR-A498) cells. The results showed increased expression of secretogranin II (SCG2, chromogranin C) in SUR-RCC cells compared to parental cells. The Cancer Genome Atlas database showed that SCG2 expression was increased in RCC compared to normal renal cells. In addition, the survival rate of the SCG2 high-expression group was significantly lower than that of the RCC low-expression group. Thus, we investigated the involvement of SCG2 in sunitinib-resistant RCC. In vitro analysis showed that migratory and invasive abilities were suppressed by SCG2 knockdown SUR cells. As SCG2 was previously reported to be associated with angiogenesis, we undertook a tube formation assay. The results showed that suppression of SCG2 inhibited angiogenesis. Furthermore, coimmunoprecipitation assays revealed a direct interaction between SCG2 and hypoxia-inducible factor 1α (HIF1α). Expression levels of VEGF-A and VEGF-C downstream of HIF1α were found to be decreased in SCG2 knockdown SUR cells. In conclusion, SCG2 could be associated with sunitinib resistance through VEGF regulation in RCC cells. These findings could lead to a better understanding of the VHL/HIF/VEGF pathway and the development of new therapeutic strategies for sunitinib-resistant RCC.

Targeting of the glutamine transporter SLC1A5 induces cellular senescence in clear cell renal cell carcinoma.

In recent years, cancer metabolism has attracted attention as a therapeutic target, and glutamine metabolism is considered one of the most important metabolic processes in cancer. Solute carrier family 1 member 5 (SLC1A5) is a sodium channel that functions as a glutamine transporter. In various cancer types, SLC1A5 gene expression is enhanced, and cancer cell growth is suppressed by inhibition of SLC1A5. However, the involvement of SLC1A5 in clear cell renal cell carcinoma (ccRCC) is unclear. Therefore, in this study, we evaluated the clinical importance of SLC1A5 in ccRCC using The Cancer Genome Atlas database. Our findings confirmed that SLC1A5 was a prognosis factor for poor survival in ccRCC. Furthermore, loss-of-function assays using small interfering RNAs or an SLC1A5 inhibitor (V9302) in human ccRCC cell lines (A498 and Caki1) showed that inhibition of SLC1A5 significantly suppressed tumor growth, invasion, and migration. Additionally, inhibition of SLC1A5 by V9302 in vivo significantly suppressed tumor growth, and the antitumor effects of SLC1A5 inhibition were related to cellular senescence. Our findings may improve our understanding of ccRCC and the development of new treatment strategies for ccRCC.

Exosomal microRNA-1 and MYO15A as a target for therapy and diagnosis in renal cell carcinoma.

Exosomes are 40-100 nm nano-sized extracellular vesicles and are receiving increasing attention as novel structures that participate in intracellular communication. We previously found that miRNA-1 (miR-1) functions as a tumor suppressor in renal cell carcinoma (RCC). In this study, we investigated the function of exosomal miR-1 and the possibility that the exosome constitutes a tumor maker in RCC. First, we established the method to collect exosomes from cell lysates and human serum by a spin column-based method. Next, we assessed exosomes using Nanosight nanoparticle tracking analysis and Western blot analysis with exosome marker CD63. We confirmed that exosomes labeled with PKH26 fused with recipient cells. Moreover, miR-1 expression was elevated in RCC cells treated with exosomes derived from miR-1-transfected cells. Functional analyses showed that exosomal miR-1 significantly inhibited cell proliferation, migration and invasion compared to control treatment. Our analyses with TCGA database of RCCs showed that miR-1 expression was significantly downregulated in clinical RCC samples compared to that in normal kidney samples, and patients with low miR-1 expression had poorer overall survival in comparison to patients with high expression. Furthermore, RNA sequence analyses showed that expression levels of several genes were altered by exposure to exosomal miR-1. The analyses with TCGA database indicated that high expression of MYO15A was associated with a poorer outcome in RCC. In addition, RT-qPCR analysis of exosomes from clinical patients' sera showed that MYO15A was significantly upregulated in RCC patients compared to that in healthy controls. This study showed that treatment with exosomal miR-1 might be an effective approach to treating RCCs. In addition, exosomal MYO15A could be a diagnostic tumor marker in RCCs.

Frontal Bone Fracture Patterns Suggesting Involvement of Optic Canal Damage.

PURPOSE: Fracture of the frontal bone can be accompanied by damage to the optic canal. The present study uses finite element analysis to identify fracture patterns, suggesting the involvement of the optic canal. METHODS: Ten finite-element skull models were generated from computer tomography data of 10 persons. Then, dynamic analyses simulating collision of a 2-cm-radius brass ball to 6 regions on the frontal bone in the 10 models were performed. Fracture patterns presented by the frontal bone in the 60 experiments were observed, and all those involving the optic canal were selected. Commonalities of the selected fracture patterns were identified. RESULTS: Fracture of the optic canal was observed in 9 of the 60 patients. In all 9 patients, fracture existed on the anterior and posterior walls of the frontal sinus and on the superior orbital wall. CONCLUSION: When the anterior and posterior walls of the frontal sinus and the superior orbital wall are all broken, the optic canal is highly likely to be involved in the damage. When this pattern is observed in emergency examination, preventive decompression of the optic nerve should be considered to avoid potential occurrence of blindness.

Utilization of a sero-muscular patch for safe wound closure after free jejunum transfer for a skin-esophageal fistula.

This paper introduces our original technique of free jejunum transfer, in which a sero-muscular patch is used to cover the jejunum. Our results demonstrate its effectiveness for touch-up surgery after esophageal leakage.

Scoring of Deformed Costal Cartilages Reduces Postoperative Pain after Nuss Procedure for Pectus Excavatum.

OBJECTIVE: The present study aims to elucidate whether or not scoring deformed cartilages reduces postoperative pain after the Nuss procedure for pectus excavatum patients. METHODS: A total of 46 pectus excavatum patients for whom the Nuss procedure was conducted were included in the study. The patients were categorized into two groups, depending on whether or not the supplementary maneuver of scoring deformed cartilages was performed in addition to the Nuss procedure. Patients for whom deformed costal cartilages were scored were categorized as the Scoring Group (n = 24); those who received no such scoring were categorized as the Non-Scoring Group (n = 22). After evaluating the maximum stresses occurring on the thoraces by means of dynamic simulation using finite element analyses, intergroup comparison of the maximum von-Mises stress values was performed. Furthermore, after quantifying postoperative pain as the frequency with which patients injected anesthetics through an epidural pain-control system within 2 postoperative days, the degree of pain was compared between the two groups. RESULTS: The maximum stresses occurring on the thorax were significantly greater for the Non-Scoring Group than for the Scoring Group; injection frequency was also greater for the Non-Scoring Group (average 4.9 times for 2 days) than for the Scoring Group (average 2.5 times for 2 days). CONCLUSION: High stresses occur due to the performance of the Nuss procedure, causing postoperative pain. The stresses can be reduced by performing supplementary scoring on deformed cartilages. Accordingly, postoperative pain is reduced.

Short-term ethanol exposure causes imbalanced neurotrophic factor allocation in the basal forebrain cholinergic system: a novel insight into understanding the initial processes of alcohol addiction.

Alcohol ingestion affects both motor and cognitive functions. One brain system that is influenced by ethanol is the basal forebrain (BF) cholinergic projection system, which projects to diverse neocortical and limbic areas. The BF is associated with memory and cognitive function. Our primary interest is the examination of how regions that receive BF cholinergic projections are influenced by short-term ethanol exposure through alterations in the mRNA levels of neurotrophic factors [nerve growth factor/TrkA, brain-derived neurotrophic factor/TrkB, and glial-derived neurotrophic factor (GDNF)/GDNF family receptor α1]. Male BALB/C mice were fed a liquid diet containing 5 % (v/v) ethanol. Pair-fed control mice were maintained on an identical liquid diet, except that the ethanol was isocalorically substituted with sucrose. Mice exhibiting signs of ethanol intoxication (stages 1-2) were used for real-time reverse transcription-polymerase chain reaction analyses. Among the BF cholinergic projection regions, decreased levels of GDNF mRNA and increased levels of TrkB mRNA were observed in the basal nucleus, and increased levels of TrkB mRNA were observed in the cerebral cortex. There were no significant alterations in the levels of expression of relevant neurotrophic factors in the septal nucleus and hippocampus. Given that neurotrophic factors function in retrograde/anterograde or autocrine/paracrine mechanisms and that BF cholinergic projection regions are neuroanatomically connected, these findings suggested that an imbalanced allocation of neurotrophic factor ligands and receptors is an initial phenomenon in alcohol addiction. The exact mechanisms underlying this phenomenon in the BF cholinergic system are unknown. However, our results provide a novel notion for the understanding of the initial processes in alcohol addiction.

Pure Orbital Floor Blow-In Fracture: An Extremely Rare Case.

A 57-year-old man presented with left diplopia on an upward gaze and ophthalmalgia after hitting the left side of his head. CT revealed a fracture on the left side of the orbital floor without orbital rim fractures and the protrusion of a small bone fragment into the orbit. Hess charts indicated markedly limited vertical movement of the left eye. Based on these findings, the patient was diagnosed with a pure orbital floor blow-in fracture (BIF). Symptoms persisted after a two-week monitoring period; therefore, the bone fragment was removed by a transcutaneous surgical approach with the assistance of a navigation system and an endoscope. Symptoms resolved after surgery, and CT and Hess examinations six months after surgery showed a good outcome. A pure BIF is rare, particularly on the orbital floor. Only a few similar case reports have been published to date, and we herein describe the surgical procedures performed and the treatment outcome of our case.

Early postnatal maternal separation causes alterations in the expression of ß3-adrenergic receptor in rat adipose tissue suggesting long-term influence on obesity.

The effects of early postnatal maternal deprivation on the biological characteristics of the adipose tissue later in life were investigated in the present study. Sprague-Dawley rats were classified as either maternal deprivation (MD) or mother-reared control (MRC) groups. MD was achieved by separating the rat pups from their mothers for 3h each day during the 10-15 postnatal days. mRNA levels of mitochondrial uncoupling protein 1 (UCP-1), ß3-adrenergic receptor (ß3-AR), and prohibitin (PHB) in the brown and white adipose tissue were determined using real-time RT-PCR analysis. UCP-1, which is mediated through ß3-AR, is closely involved in the energy metabolism and expenditure. PHB is highly expressed in the proliferating tissues/cells. At 10 weeks of age, the body weight of the MRC and MD rats was similar. However, the levels of the key molecules in the adipose tissue were substantially altered. There was a significant increase in the expression of PHB mRNA in the white adipose tissue, while the ß3-AR mRNA expression decreased significantly, and the UCP-1 mRNA expression remained unchanged in the brown adipose tissue. Given that these molecules influence the mitochondrial metabolism, our study indicates that early postnatal maternal deprivation can influence the fate of adipose tissue proliferation, presumably leading to obesity later in life.

Effects of flow patterns and hemodynamic force on vascular endothelium in the temporary arteriovenous shunt loop in rabbits.

The purpose of this study was to investigate whether there is a risk of thrombosis in the temporary arteriovenous shunt loop (TAVSL). The authors established a TAVSL model in the rabbit. Experimental groups were divided into non-heparin treated and heparin treated. The maximum blood flow volume, blood viscosity, and radius of curvature were measured, and the Reynolds number and the sheer stress were calculated. Computational fluid dynamics (CFD) was used to predict the flow pattern in the TAVSL, and these predicted data were compared with histological results. Early occlusion was noted in 70% (7/10) of the non-heparin-treated group and 22% (2/9) of the heparin-treated group. CFD analysis predicted a high shear stress at the arterial anastomosis region and the outer luminal surface of the curved section. The intimal structure at the luminal surface of the curved section was extensively lost histologically. In the patent group, severe stenosis of the lumen was noted at the apex of the loop due to an organized thrombus. Thus, thrombosis is likely to occur in the TAVSL due to endothelium injury caused by high shear stress, and this results in the formation of white thrombi at an early stage and an organized thrombus at a late stage.

Separation of the seventh costal-sternal junction-A new technique to improve outcomes for the Nuss procedure for pectus excavatum.

BACKGROUND: In the standard Nuss procedure for pectus excavatum, the costal arch is often elevated together with the sternum, resulting in unevenness of the lower part of the thorax. This complication is commonly called rib flaring. This paper presents a technique to avoid rib flaring and evaluates its effectiveness. MATERIALS AND METHODS: In our technique, a part of the seventh costal cartilage is removed, disconnecting the costal arch from the sternum. The effectiveness of this technique was evaluated in a retrospective clinical study of 63 pectus excavatum patients who were randomly collected and were divided into two groups. One group-defined as the Standard Group-includes 27 patients (29.8 ± 6.5SD y/o) on whom standard Nuss procedure was conducted; the other group-defined as the Separation Group-includes 36 patients (31.8 ± 6.1SD y/o) on whom the cartilage removal was conducted in addition to the standard Nuss procedure. The degree of postoperative costal-arch elevation was defined as ECA (Elevation of Costal Arch) and was compared between the two groups. RESULTS: ECA was significantly greater for the Standard Group (10.2 ± 3.3SD mm) than for the Separation Group (-1.1 ± 3.42SDmm). CONCLUSION: Postoperative protrusion of the costal arch is prevented by the separation of the seventh costal cartilage from the sternum. Our original technique is a useful option for the treatment of pectus excavatum.

Characterization and treatment of gemcitabine- and cisplatin-resistant bladder cancer cells with a pan-RAS inhibitor.

Combination chemotherapy with gemcitabine and cisplatin (GC) is recommended as the primary treatment for advanced bladder cancer (BC). However, the benefits of this approach are limited owing to the acquisition of drug resistance. Here, we found that gemcitabine-resistant and cisplatin-resistant BCs do not exhibit cross-resistance, and that these BCs exhibit different mRNA patterns, as revealed using RNA sequence analysis. To overcome drug resistance, we used the newly developed pan-RAS inhibitor Compound 3144. Compound 3144 inhibited cell viability through suppression of RAS-dependent signaling in gemcitabine- and cisplatin-resistant BCs. RNA sequencing revealed that several genes and pathways, particularly those related to the cell cycle, were significantly downregulated in Compound 3144-treated BCs. These findings provide insights into potential therapeutic strategies for treating BC.

Morphological evidence of an altered process of synaptic transcytosis in adult rats exposed to ethanol.

AIMS: The effects of ethanol exposure on synaptic structure were investigated in the nucleus of solitary tract (NST) in rats, using the horse-radish peroxidase (HRP) method. METHODS: Eight-week-old experimental rats were allowed free access to a liquid diet containing ethanol for 3 weeks, while controls were given an isocaloric diet. Some of the control and experimental animals were given an injection of wheat germ agglutinin conjugated with HRP (WGA-HRP) into the vagus nerve toward the end of the treatment period. After the treatment, the neuropil region of the NST was examined under an electron microscope. RESULTS: We observed that a few terminals were characterized by deep indentation of axodendritic membranes into the post-synaptic neurons. This appeared to be similar to that commonly seen in exocrine glands. Interestingly, the indented portion often contained various sizes of vacuoles and flattened cisternae. HRP-reaction product (RP) transported to terminals was recognized easily as an electron-dense lysosomal substance when lead citrate staining was omitted. Terminals containing HRP-RP also revealed quite a similar structure with indentation of axodendritic membranes as described earlier. The results are considered to confirm that terminals forming 'apocrine-like structures' observed in the ethanol-fed animals with no injection of WGA-HRP originate from afferent fibers of the vagus nerve. CONCLUSION: The present study suggests the possibility that the alteration of the synaptic structure induced by ethanol exposure can lead to the neuronal transcytosis of materials including proteins which is different from the normal vesicular exocytosis involved in chemical synaptic transmission.

How Partial Skull Defect Affects Vulnerability of the Skull in Traumatic Situations: A Biomechanical Study.

Background: Part of the skull can be lost due to neurosurgical diseases or trauma. Skulls with partial defects can develop different fracture patterns from those of intact skulls. This study aims to clarify the differences. Methods: A 3-dimensional skull model was produced by referring to the computer-tomography data of a 23-year-old intact male volunteer. We defined the model as Intact Model. Another model was produced by removing part of the frontal bone, which was defined as Defect Model. Dynamic simulations of impacts were performed varying the site and direction of impact. Fracture patterns caused by the impacts were calculated using dynamic analysis software (LS-DYNA; Livermore Software Technology Corp.) and were compared between the intact model and defect model. Results: When Defect Model was impacted, fracture involved wider areas than when Intact Model was impacted. This finding was observed not only when Defect Model was impacted on its defect side but also when it was impacted on its intact side. Conclusions: When a skull carrying a defect on one side is impacted, serious fracture occurs even when the non-defect side is impacted, meaning that a skull with a defect is vulnerable to impacts on the non-defect side. This finding should be taken into consideration in deciding indications of skull defect reconstruction.

microRNA-99a-5p induces cellular senescence in gemcitabine-resistant bladder cancer by targeting SMARCD1.

Patients with advanced bladder cancer are generally treated with a combination of chemotherapeutics, including gemcitabine, but the effect is limited due to acquisition of drug resistance. Thus, in this study, we investigated the mechanism of gemcitabine resistance. First, gemcitabine-resistant cells were established and resistance confirmed in vitro and in vivo. Small RNA sequencing analyses were performed to search for miRNAs involved in gemcitabine resistance. miR-99a-5p, selected as a candidate miRNA, was downregulated compared to its parental cells. In gain-of-function studies, miR-99a-5p inhibited cell viabilities and restored sensitivity to gemcitabine. RNA sequencing analysis was performed to find the target gene of miR-99a-5p. SMARCD1 was selected as a candidate gene. Dual-luciferase reporter assays showed that miR-99a-5p directly regulated SMARCD1. Loss-of-function studies conducted with si-RNAs revealed suppression of cell functions and restoration of gemcitabine sensitivity. miR-99a-5p overexpression and SMARCD1 knockdown also suppressed gemcitabine-resistant cells in vivo. Furthermore, ß-galactosidase staining showed that miR-99a-5p induction and SMARCD1 suppression contributed to cellular senescence. In summary, tumor-suppressive miR-99a-5p induced cellular senescence in gemcitabine-resistant bladder cancer cells by targeting SMARCD1.

Separation patterns of orbital wall and risk of optic canal injury in Le Fort 3 osteotomy.

PURPOSE: The authors hypothesized that the risks of optic canal injury in down-fracturing after Le Fort 3 osteotomy vary depending on the separation patterns of the orbital walls. This study verifies this hypothesis using biomechanical simulation. METHODS: Ten finite-element skull models were produced using computer tomography data from ten persons. These models were modified to simulate Le Fort 3 osteotomy models by removing junctions between the neurocranium and facial cranium. The separation of the orbital wall was performed in four differing ways. In Type 1, all walls were completely separated. In Type 2, only the lateral wall was separated. In Type 3, the inferior wall was left unseparated. In Type 4, the lateral wall was left unseparated. Biomechanical simulation of down-fracturing was performed on the resulting 40 models. By observing irregular fractures occurring inside the orbit, the rate of optic canal involvement was evaluated for each of the four orbital-wall separation patterns. RESULTS: The rates of optic canal involvement were: Type 1 (0/10), Type 2 (0/10), Type 3 (0/10), and Type 4 (4/10). CONCLUSION: When the lateral wall is incompletely separated in Le Fort 3 osteotomy, irregular fracture can develop inside the orbit and involve the optic canal during the down-fracturing process. Hence, the lateral orbital wall should be completely separated to avoid potential blindness due to optic canal injury.

Biomechanical analysis of likelihood of optic canal damage in peri-orbital fracture.

PURPOSE: Detection of optic canal fractures is often difficult because of the subtleness of the fracture. If we could clarify impact on which region around the orbit is likely to accompany the fracture of the optic canal, the knowledge should be useful to make early diagnosis of optic canal fractures. The present study was conducted to elucidate this issue. METHODS: Ten finite element models were produced simulating the skulls of ten humans (8 males and 2 females; 43.8 ± 10.2 y/o). The peri-orbital area of each of the ten models was divided into eight regions in a clockwise fashion per 45 degrees. These regions were defined as Superior-Medial (0-45 degrees), Medial-Superior (45-90 degrees), Medial-Inferior (90 to 135 degrees), Inferior-Medial (135 to 180 degrees), Inferior-Lateral (180-225 degrees), Lateral-Inferior (225 to 270 degrees), Lateral-Superior (270-315 degrees), and Superior-Lateral regions (315-360 degrees), respectively. Dynamic simulation of applying traumatic energy on each of these regions was conducted. Resultant fracture patterns were evaluated using finite element analyses. Thereafter, frequencies of fracture involvement of the optic canal were evaluated for each of the eight regions. RESULTS: The involvement of the optic canal was most frequent for the Superior-Medial region (7/10), followed by the Medial-Superior region (5/10). CONCLUSION: Optic canal fracture is likely to occur when the area between the supra-orbital notch and the medial canthus are strongly impacted. When evident fracture or serious damage of soft tissue is observed in this area, occurrence of optic canal fracture should be suspected.

Normobaric oxygen therapy increases cartilage survival ratio in auricular composite grafting in rat models.

PURPOSE: This study aims to clarify whether normobaric oxygen therapy improves the survival of auricular composite grafts in rats. METHODS: For 10 male SD rats, 1.5 cm2 composite grafts were harvested from bilateral ear regions including whole auricles. The harvested grafts were transferred caudally and sutured there. The 10 rats were randomly divided into two groups and kept for 21 days in two different circumstances. The first group (Control group: five rats carrying 10 grafts) was kept in room air (20% oxygen) throughout the 21 days, and the second group-named NBO (normobaric oxygen) group (five rats carrying 10 grafts)-was kept in normobaric 60% oxygen for 3 days and then in room air for 18 days. All the 10 rats were sacrificed on the 21st day. Surviving areas of the grafts and the height of the surviving auricular cartilage were examined for statistical comparison of the two groups. Furthermore, the conditions of chondrogenesis occurring around the perichondrium were compared between the two groups. RESULTS: Surviving areas did not present statistically significant differences between the two groups. The height of surviving cartilage was significantly greater for the NBO group (2610 ± 170 SD µm) than that for the Control group (1720 ± 190 SD µm). Chondrogenesis occurred at positions more distant from the recipient bed in the NBO group than that in the Control group. CONCLUSION: Normobaric oxygen therapy increases the thickness of surviving cartilage in auricular composite grafting in rats, thus suggesting that NBO therapy may also be effective in composite grafting for humans.

Thoracic outlet syndrome after the Nuss procedure for pectus excavatum: Is it a rare complication?

OBJECTIVE: The present study aims to elucidate the frequency of thoracic outlet syndrome after the Nuss procedure for pectus excavatum and the conditions in which thoracic outlet syndrome is likely to develop. METHODS: A retrospective study including 85 pectus excavatum patients (58 males and 27 females) was conducted. Thoracic outlet syndrome was defined as a condition in which the patient has numbness, lassitude, or pain of the upper limbs at rest or during motion of the upper limbs. The frequency of the thus-defined thoracic outlet syndrome was evaluated in 85 patients. Age, sex, Haller indices, and the positions of the correction bars were compared between the patients who developed thoracic outlet syndrome and those who did not. RESULTS: Preadolescent patients (18 out of 85) did not develop postoperative thoracic outlet syndrome. In total, 15.2% of adult male patients (7 out of 46) and 33% of adult female patients (7 out of 21) developed postoperative thoracic outlet syndrome. For both male and female groups, Haller indices were significantly greater for patients who had postoperative thoracic outlet syndrome than for those who did not. Correction bars were generally placed at higher intercostal spaces in patients who developed postoperative thoracic outlet syndrome than in those who did not. CONCLUSION: A considerable percentage of adult patients develop thoracic outlet syndrome after the Nuss procedure for pectus excavatum. Maturity of the thoracic wall, femininity, severity of the deformity (represented by greater Haller indices), and placement of correction bars at superior intercostal spaces are risk factors for postoperative thoracic outlet syndrome.