Evaluation of the Angle Between the Long Axis of the Femoral Anterior Cruciate Ligament Footprint and Bony Morphology of the Knee: A Cadaveric Descriptive Study.

Purpose: There have been numerous studies of the anterior cruciate ligament (ACL) anatomy, but few have focused on the long axis angle of the femoral ACL footprint. This study investigated the angle between the long axis of the femoral ACL footprint and the bony morphology of the knee. Methods: This study is a cadaveric descriptive study. Thirty non-paired formalin-fixed knees of Japanese cadavers were used. Anteromedial (AM) and posterolateral (PL) bundles were identified according to the tension pattern differences during the complete range of motion of the knee. In the ACL femoral footprint, there is a fold between the mid-substance insertion site and fan-like extension fibers. After identifying AM and PL bundles of mid-substance fibers, the mid-substance and fan-like extension fibers were divided into those bundles and stained. We defined the line passing through the center of the AM and PL bundles as the long axis of the ACL. The center points of each of the four areas and the angle between the long axis of the ACL and the bony morphology of the knee were calculated using Image J software. Results: The mean angle between the axis of the femoral shaft and the long axis of the ACL mid-substance insertion was 28.8 ± 12.2 degrees. The mean angle between the Blumensaat line and the long axis of the mid-substance was 54.2 ± 13.5 degrees. Conclusion: The mean angle between the axis of the femoral shaft and the long axis of the femoral ACL footprint was approximately 29 degrees. There is a wide variation in the long axis of the femoral ACL footprint. To achieve better clinical results through a more anatomically accurate reconstruction, it can be beneficial to replicate the ACL femoral footprint along its native long axis.

SCD1 inhibition enhances the effector functions of CD8+ T cells via ACAT1-dependent reduction of esterified cholesterol.

We previously reported that the inhibition of stearoyl-CoA desaturase 1 (SCD1) enhances the antitumor function of CD8+ T cells indirectly via restoring production of DC recruiting chemokines by cancer cells and subsequent induction of antitumor CD8+ T cells. In this study, we investigated the molecular mechanism of direct enhancing effects of SCD1 inhibitors on CD8+ T cells. In vitro treatment of CD8+ T cells with SCD1 inhibitors enhanced IFN-γ production and cytotoxic activity of T cells along with decreased oleic acid and esterified cholesterol, which is generated by cholesterol esterase, acetyl-CoA acetyltransferase 1 (ACAT1), in CD8+ T cells. The addition of oleic acid or cholesteryl oleate reversed the enhanced functions of CD8+ T cells treated with SCD1 inhibitors. Systemic administration of SCD1 inhibitor to MCA205 tumor-bearing mice enhanced IFN-γ production of tumor-infiltrating CD8+ T cells, in which oleic acid and esterified cholesterol, but not cholesterol, were decreased. These results indicated that SCD1 suppressed effector functions of CD8+ T cells through the increased esterified cholesterol in an ACAT1-dependent manner, and SCD1 inhibition enhanced T cell activity directly through decreased esterified cholesterol. Finally, SCD1 inhibitors or ACAT1 inhibitors synergistically enhanced the antitumor effects of anti-PD-1 antibody therapy or CAR-T cell therapy in mouse tumor models. Therefore, the SCD1-ACAT1 axis is regulating effector functions of CD8+ T cells, and SCD1 inhibitors, and ACAT1 inhibitors are attractive drugs for cancer immunotherapy.

Serum Free Fatty Acid Changes Caused by High Expression of Stearoyl-CoA Desaturase 1 in Tumor Tissues Are Early Diagnostic Markers for Ovarian Cancer.

Ovarian cancer has a poor prognosis and is difficult to detect in early stages. Therefore, developing new diagnostic markers for early-stage ovarian cancer is critical. Here, we developed a diagnostic marker for early-stage ovarian cancer on the basis of fatty acid metabolism characteristics of cancer cells. The expression of various fatty acid metabolizing enzymes such as stearoyl-CoA desaturase 1 (SCD1) was altered in early-stage ovarian cancer tissue compared with that in normal ovarian tissue. Changes in the expression of fatty acid metabolizing enzymes, particularly SCD1, in cancer tissues were found to alter concentrations of multiple free fatty acids (FFA) in serum. We were the first to show that fatty acid metabolic characteristics in tissues are related to the FFA composition of serum. Surprisingly, patients with stage I/II ovarian cancer also showed significant changes in serum levels of eight FFAs, which can be early diagnostic markers. Finally, using statistical analysis, an optimal early diagnostic model combining oleic and arachidic acid levels, fatty acids associated with SCD1, was established and confirmed to have higher diagnostic power than CA125, regardless of histology. Thus, our newly developed diagnostic model using serum FFAs may be a powerful tool for the noninvasive early detection of ovarian cancer. SIGNIFICANCE: Measurement of serum FFA levels by changes in the expression of fatty acid metabolizing enzymes in tumor tissue would allow early detection of ovarian cancer. In particular, the SCD1-associated FFAs, oleic and arachidic acid, would be powerful new screening tools for early-stage ovarian cancer.

Macrophage depletion using clodronate liposomes reveals latent dysfunction of the hematopoietic microenvironment associated with persistently imbalanced M1/M2 macrophage polarization in a mouse model of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH), a hyperinflammatory syndrome, is caused by the incessant activation of lymphocytes and macrophages, resulting in damage to organs, including hematopoietic organs. Recently, we demonstrated that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated (SAMP1/TA-1) mice but not in senescence-resistant control (SAMR1) mice. Hematopoietic failure in LPS-treated SAMP1/TA-1 mice was attributed to hematopoietic microenvironment dysfunction, concomitant with severely imbalanced M1 and M2 macrophage polarization. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment. Clodronate liposomes are useful tools for in vivo macrophage depletion. In this study, we depleted macrophages using clodronate liposomes to determine their role in the hematopoietic microenvironment in SAMP1/TA-1 and SAMR1 mice. Under clodronate liposome treatment, the response between SAMR1 and SAMP1/TA-1 mice differed as follows: (1) increase in the number of activated M1 and M2 macrophages derived from newly generated macrophages and M2-dominant and imbalanced M1 and M2 macrophage polarization in the BM and spleen; (2) severe anemia and thrombocytopenia; (3) high mortality rate; (4) decrease in erythroid progenitors and B cell progenitors in the BM; and (5) decrease in the mRNA expression of erythroid-positive regulators such as erythropoietin and increase in that of erythroid- and B lymphoid-negative regulators such as interferon-γ in the BM. Depletion of residual macrophages in SAMP1/TA-1 mice impaired hematopoietic homeostasis, particularly erythropoiesis and B lymphopoiesis, owing to functional impairment of the hematopoietic microenvironment accompanied by persistently imbalanced M1/M2 polarization. Thus, macrophages play a vital role in regulating the hematopoietic microenvironment to maintain homeostasis.

Imbalanced M1 and M2 Macrophage Polarization in Bone Marrow Provokes Impairment of the Hematopoietic Microenvironment in a Mouse Model of Hemophagocytic Lymphohistiocytosis.

Lipopolysaccharide (LPS) treatment induced hemophagocytic lymphohistiocytosis in senescence-accelerated mice (SAMP1/TA-1), but not in senescence-resistant control mice (SAMR1). SAMP1/TA-1 treated with LPS exhibited functional impairment of the hematopoietic microenvironment, which disrupted the dynamics of hematopoiesis. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment, which regulates hematopoiesis. Qualitative and quantitative changes in activated macrophages in LPS-treated SAMP1/TA-1 are thought to contribute to the functional deterioration of the hematopoietic microenvironment. Thus, we examined the polarization of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, and the dynamics of macrophage production in the BM of SAMP1/TA-1 and SAMR1 after LPS treatment. After LPS treatment, the proportions of M1 and M2 macrophages and the numbers of macrophage progenitor (CFU-M) cells increased in both SAMP1/TA-1 and SAMR1. However, compared to the SAMR1, the increase in the M1 macrophage proportion was prolonged, and the increase in the M2 macrophage proportion was delayed. The increase in the number of CFU-M cells was prolonged in SAMP1/TA-1 after LPS treatment. In addition, the levels of transcripts encoding an M1 macrophage-inducing cytokine (interferon-γ) and macrophage colony-stimulating factor were markedly increased, and the increases in the levels of transcripts encoding M2 macrophage-inducing cytokines (interleukin (IL)-4, IL-10, and IL-13) were delayed in SAMP1/TA-1 when compared to SAMR1. Our results suggest that LPS treatment led to the severely imbalanced polarization of activated M1/M2 macrophages accompanied by a prolonged increase in macrophage production in the BM of SAMP1/TA-1, which led to the impairment of the hematopoietic microenvironment, and disrupted the dynamics of hematopoiesis.

Combination of serum 5-S-cysteinyldopa, melanoma inhibitory activity and IL-8 improves the diagnostic accuracy of malignant melanoma compared with individual markers.

Early diagnosis of malignant melanoma is critical for effective treatment and reduced patient mortality. However, current clinical and histological variables show limited accuracy in diagnosis. Serum or urine level of 5-S-cysteinyldopa (5-S-CD) is a commonly used melanoma biomarker in Japan owing to its increased sensitivity compared with other melanoma markers. However, its use as a diagnostic marker has shown some limitations. Therefore, here we examined the combination of 5-S-CD with melanoma inhibitory activity, which showed sensitivity in detecting melanoma comparable with that of 5-S-CD, and interleukin-8, a cytokine linked with melanoma progression, in a cohort of Japanese patients with melanoma. Our results revealed that the triple combination of 5-S-CD, melanoma inhibitory activity, and interleukin-8 showed high diagnostic accuracy in detecting melanoma compared with each of the individual factors. Importantly, the triple marker showed specificity and utility in detecting early-stage melanoma. Our results suggest the utility of the triple marker as a diagnostic biomarker for melanoma patients.

Short-term high-fat and high-carbohydrate diets increase susceptibility to liver injury by inducing hepatic procoagulant and proinflammatory conditions with different balances.

OBJECTIVES: High-fat diet (HFD) and high-carbohydrate diet (HCD) are strongly linked to nonalcoholic fatty liver disease, a hepatic manifestation of metabolic syndrome. The mechanism of pathologic progression from nonalcoholic fatty liver disease to nonalcoholic steatohepatitis, which is a more severe form associated with inflammation and fibrosis, remains poorly understood. Thus, the aim of this study was to investigate and compare the inflammatory and coagulative state of the liver in short-term HFD- or HCD-fed mice with acute liver injury induced by concanavalin A (Con A). METHODS: Histopathologic evaluation, real-time polymerase chain reaction, and immunohistochemical evaluation were performed on the liver of mice fed HFDs and HCDs for 4 d before and after Con A administration. RESULTS: The liver of the HFD-fed mice had larger fibrinogen/fibrin depositions than those fed the HCD. HCD induced the expression of the proinflammatory cytokine tumor necrosis factor-α in the liver. Moreover, the expression of proinflammatory cytokines and chemokines was further enhanced after Con A stimulation in HCD (e.g., interleukin-1α, interleukin-6 at 1 h), with a strong tendency for inflammatory cell infiltration also found (24 h). CONCLUSIONS: Short-term HCD and HFD increased susceptibility to liver injury. HCD tended to induce more intense inflammation, whereas HFD tended to induce more intense hypercoagulation, suggesting that HCD and HFD may have different mechanisms of pathologic progression to nonalcoholic steatohepatitis.

Specific acupuncture stimulation of Shenshu (BL23) affects sympathetic nervous activity-associated plasma renin concentration changes.

OBJECTIVE: To examine whether specific stimulation of Shenshu (BL23) affects sympathetic nervous activity (SNA)-associated plasma renin concentration (PRC). METHODS: Eight healthy volunteers participated in three pattern conditions in random order: control (Cont), stimulation of Shenshu (BL23), and stimulation of sham point (Sham). All participants were initially in the supine position for > 60 min, and then remained in the standing position during the experimental procedure to increase SNA. An electrocardiogram was used to calculate low frequency/high frequency (LF/HF) ratio; blood was collected to analyze PRC. RESULTS: The LF/HF ratio was significantly increased in the standing position when compared with the supine position ( 0.01). There was no difference in LF/HF ratio during or after stimulation of Shenshu (BL23) in the standing position when compared with before the stimulation in the supine position; however, the LF/HF ratio was significantly increased in Cont and Sham conditions ( 0.01). There was no difference in PRC after stimulation of Shenshu (BL23) in the standing position when compared with before the stimulation in the supine position; however, there was a significant increase in PRC in the Cont and Sham conditions (Cont 0.05, Sham 0.01). CONCLUSION: Our results demonstrated that specific acupuncture stimulation of Shenshu (BL23) in the standing position decreased SNA-associated PRC, which was not observed during acupuncture stimulation of the sham point.

Efficacy of urea solution reperfusion to a formalin-embalmed cadaver for surgical skills training.

Formaldehyde has been traditionally used for embalming human cadavers for gross anatomy education and surgical skills training. However, exposure to formaldehyde negatively affects human health. This study aimed to assess the efficacy of reperfusing urea solution to a formalin-embalmed cadaver for surgical skills training and then investigate the cadaver's tissue elasticity alteration after being soaked into the urea solution. Twelve surgeons evaluated the similarity of tissue characteristics between the cadaver (embalmed by formalin solution and reperfused by urea solution) and a living human body. Furthermore, the tissue formaldehyde content and mechanical properties of the formalin-fixated femoral skin and artery specimens with or without soaking into urea solution were measured. Results showed that the tactile assessment, skin incision, vessel ligation and suture, and decollement were better and more useful in the cadaver reperfused by urea solution than in the cadaver merely fixated by formalin solution. In the urea-reperfused cadaver, the volatilized, or tissue formaldehyde levels declined. The stiffness and Young's modulus of the femoral skin and artery were also lower in the specimen than in the mere formalin-fixated specimen. In conclusion, reperfusion of urea solution to the formalin-fixated cadaver makes anatomical education and surgical skills training more efficient with fewer requirements for cadaver management.

GIT1 is an untolerized autoantigen involved in immunologic disturbance of spermatogenesis.

Male haploid cells, spermatids and spermatozoa, that appear after the establishment of immune tolerance express novel cell surface and intracellular proteins that can be recognized as foreign antigens by the self-immune system. However, these germ cells do not normally evoke a pathological immune response. The immune-privileged micro-circumstance in testis involving the blood-testis-barrier formed by Sertoli cells protects these germ cells from autoimmune attack. We recently found that immunization with heat shock protein family A member 4-like (HSPA4L), one of the new differentiation antigens of haploid cells, induced experimental autoimmune orchitis (EAO) in A/J male mice. In this study, we focused on G protein-coupled receptor kinase interacting protein-1 (GIT1), another haploid cell-specific differentiation antigen, to investigate whether GIT1 is a target autoantigen for EAO induction. GIT1 emulsified with complete Freund's adjuvant was injected subcutaneously into the mice inguinal region once on day 0 and again on day 14, and the optimum condition of EAO induction was determined. Mice immunized with 200 µg GIT1 showed significantly higher incidence of EAO than that of immunization with other concentrations. In particular, significant lymphocytic inflammation and extensive aspermatogenesis were observed in these mice at 120 days after the first immunization. These findings indicate that GIT1 is also a target antigen that induces EAO, like HSPA4L.

The effect of simulation-based education before a cadaver dissection course.

Although the methods for medical education continue to evolve due to the development of medicines, the cadaver dissection course still plays a fundamental role. The cadaver dissection course allows students to learn to handle instruments correctly while actively exploring three-dimensional anatomy. However, dissection comes with the risk of accidental injury. In recent years, the number of classes offered for the cadaver dissection course has decreased while the amount of knowledge required in clinical medicine has increased. Simulation-based education (SBE) has been proven to be an effective educational method that enhances the development of practical skills by integrating learners' knowledge and skills. This study aimed to investigate the effect of SBE as a preparatory education course when taken prior to a medical student's enrollment in the cadaver dissection course. In the present study, an SBE assuming practical cadaver dissection course was performed in the Clinical Simulation Center. The frequency of injury rates per 1000 h of cadaver dissection course was significantly less in 2017 and 2018 compared to that in 2016. Two years after the implementation of the SBE, average student self-efficacy scores and written examination scores significantly increased, whereas self-contentment scores were relatively unchanged. The results showed that the implementation of SBE decreased the incidence of injuries and improved students' overall self-efficacy scores and increased acquisition of knowledge evident on written examination score. Therefore, SBE as a preparatory education course may effectively promote the combined development of dissection skills and anatomical knowledge in the subsequent fundamental cadaver dissection course.

Anorectal Transplantation: The First Long-term Success in a Canine Model.

OBJECTIVE: Anorectal transplantation is a challenging procedure but a promising option for patients with weakened or completely absent anorectal function. SUMMARY BACKGROUND DATA: We constructed a canine model of anorectal transplantation, evaluated the long-term outcomes, and controlled rejection and infection in allotransplantation. METHODS: In the pudendal nerve function study, 6 dogs were randomly divided into 2 groups, transection and anastomosis, and were compared with a control using anorectal manometry, electromyography, and histological examination. In the anorectal transplantation model, 4 dogs were assigned to 4 groups: autotransplant, allotransplant with immunosuppression, allotransplant without immunosuppression, and normal control. Long-term function was evaluated by defecography, videography, and histological examination. RESULTS: In the pudendal nerve function study, anorectal manometry indicated that the anastomosis group recovered partial function 6 months postoperatively. Microscopically, the pudendal nerve and the sphincter muscle regenerated in the anastomosis group. Anorectal transplantation was technically successful with a 3-stage operation: colostomy preparation, anorectal transplantation, and stoma closure. The dog who underwent allotransplantation and immunosuppression had 2 episodes of mild rejection, which were reversed with methylprednisolone and tacrolimus. The dog who underwent allotransplantation without immunosuppression had a severe acute rejection that resulted in graft necrosis. Successful dogs had full defecation control at the end of the study. CONCLUSIONS: We describe the critical role of the pudendal nerve in anorectal function and the first long-term success with anorectal transplantation in a canine model. This report is a proof-of-concept study for anorectal transplantation as a treatment for patients with an ostomy because of anorectal dysfunction.

Age-related exacerbation of hematopoietic organ damage induced by systemic hyper-inflammation in senescence-accelerated mice.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyper-inflammatory disorder. The mortality of HLH is higher in the elderly than in young adults. Senescence-accelerated mice (SAMP1/TA-1) exhibit characteristic accelerated aging after 30 weeks of age, and HLH-like features, including hematopoietic organ damage, are seen after lipopolysaccharide (LPS) treatment. Thus, SAMP1/TA-1 is a useful model of hematological pathophysiology in the elderly with HLH. In this study, dosing of SAMP1/TA-1 mice with LPS revealed that the suppression of myelopoiesis and B-lymphopoiesis was more severe in aged mice than in young mice. The bone marrow (BM) expression of genes encoding positive regulators of myelopoiesis (G-CSF, GM-CSF, and IL-6) and of those encoding negative regulators of B cell lymphopoiesis (TNF-α) increased in both groups, while the expression of genes encoding positive-regulators of B cell lymphopoiesis (IL-7, SDF-1, and SCF) decreased. The expression of the GM-CSF-encoding transcript was lower in aged mice than in young animals. The production of GM-CSF by cultured stromal cells after LPS treatment was also lower in aged mice than in young mice. The accumulation of the TNF-α-encoding transcript and the depletion of the IL-7-encoding transcript were prolonged in aged mice compared to young animals. LPS dosing led to a prolonged increase in the proportion of BM M1 macrophages in aged mice compared to young animals. The expression of the gene encoding p16INK4a and the proportion of ß-galactosidase- and phosphorylated ribosomal protein S6-positive cells were increased in cultured stromal cells from aged mice compared to those from young animals, while the proportion of Ki67-positive cells was decreased in stromal cells from aged mice. Thus, age-related deterioration of stromal cells probably causes the suppression of hematopoiesis in aged mice. This age-related latent organ dysfunction may be exacerbated in elderly people with HLH, resulting in poor prognosis.

Roles of glomerular endothelial hyaluronan in the development of proteinuria.

Vascular endothelial cells are covered with glycocalyx comprising heparan sulfate, hyaluronan, chondroitin sulfate, and associated proteins. Glomerular endothelial glycocalyx is involved in protecting against induction of proteinuria and structural damage, but the specific components in glycocalyx that represent therapeutic targets remain unclear. Anti-vascular endothelial growth factor (VEGF) therapy is associated with an increased risk of glomerular endothelial injury. This study investigated whether hyaluronan could provide a therapeutic target to protect against proteinuria. We conducted ex vivo and in vivo experiments to explore the effects of degrading glomerular hyaluronan by administering hyaluronidase and of supplementation with hyaluronan. We investigated hyaluronan expression using biotin-labeled hyaluronan-binding protein (HABP) in human kidney specimens or serum hyaluronan in endothelial injuries under inhibition of VEGF signaling. We directly demonstrated hyaluronan in glomerular endothelial layers using HABP staining. Ex vivo and in vivo experiments showed the development of proteinuria after digestion of hyaluronan in glomerular capillaries. Supplementation with hyaluronan after hyaluronidase treatment suppressed proteinuria. Mice in the in vivo study developed albuminuria after intraperitoneal injection of hyaluronidase with decreased glomerular hyaluronan and increased serum hyaluronan. In human kidneys with endothelial cell dysfunction and proteinuria due to inhibition of VEGF, glomerular expression of hyaluronan was reduced even in normal-appearing glomeruli. Serum hyaluronan levels were elevated in patients with pre-eclampsia with VEGF signaling inhibition. Our data suggest that hyaluronan itself plays crucial roles in preventing proteinuria and preserving the integrity of endothelial cells. Hyaluronan could provide a therapeutic target for preventing glomerular endothelial glycocalyx damage, including VEGF signaling inhibition.

Efficacy of Methotrexate on Rat Knee Osteoarthritis Induced by Monosodium Iodoacetate.

OBJECTIVE: To explore whether methotrexate (MTX) prevents joint destruction and improves pain-related behaviors in the acute phase of knee osteoarthritis (OA) induced by monosodium iodoacetate (MIA) in a rat model. METHODS: Twenty of 25 male Wistar rats (10-14 weeks old) received 3 mg MIA via intra-articular injection into their right knee and were then administered a vehicle control (n=10) or 3 mg/kg MTX orally weekly (n=10). We assessed differences in pain-related behavior, spontaneous lifting behavior, micro-computed tomography (CT), histopathology, and expression of pain- and inflammatory-related genes using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) between the two groups for 4 weeks. Five rats were used as untreated controls to assess pain- and inflammatory-related mRNA expression in the dorsal root ganglia (DRG) and knee joints using RT-qPCR. RESULTS: Joint destruction and mechanical hyperalgesia were observed in the vehicle group. Decreases in mechanical pain thresholds for the knee joint and calf muscles were improved after MTX administration; however, joint damage assessed by micro-CT and histopathology was not significantly inhibited by MTX administration, while upregulation levels of transient receptor potential cation channel, subfamily V, member 1 (TRPV-1) (P<0.01) and brain-derived neurotrophic factor (BDNF) (P=0.02) mRNA in the DRG and nerve growth factor NGF mRNA (P=0.03) in the affected knee joints were significantly suppressed in the MTX group compared with the vehicle group at week 4. CONCLUSION: Our results imply that MTX administration improves pain-related behaviors and suppresses expression of pain-related mRNAs in the DRG and knee joint; however, MTX is not expected to prevent cartilage degeneration in MIA-induced OA in rat knee.

Sulfide catabolism ameliorates hypoxic brain injury.

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

Heat shock protein A4L is a potent autoantigen for testicular autoimmunity in mice.

Experimental autoimmune orchitis (EAO) may be used as a model to investigate immunological infertility in men. Murine EAO is induced via immunization with auto-immunogenic antigens (AIAgs) from testicular germ cells (TGCs). CD4 + T cells play a crucial role in EAO induction. However, whether AIAgs induce an immune response remains unclear. We aimed to identify self-antigens that induce EAO by screening a phage display library of random TGC peptides using IgG from EAO-induced A/J mice. Twenty TGC-specific AIAgs were detected, and G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) and heat shock protein A4L (HSPA4L) were identified as candidate AIAgs that induce EAO. Immunization with GIT1 or HSPA4L, emulsified in complete Freund's adjuvant, resulted in 66 % or 100 % incidence of EAO, respectively, indicating that HSPA4L is a most potent AIAg that induces EAO in mice. These findings may expectedly help improve the diagnostic procedures and treatment of immunological infertility in men.

A rare third head of the biceps femoris in the posterior thigh.

The occurrence of a third head of the biceps femoris is very rare. We encountered the case of a 90-year-old Japanese male cadaver with a third head of the biceps femoris in the posterior aspect of the thigh during dissection at Aichi Medical University in 2016. It originated from the proximal part of the femur and fused with the muscle belly between the long and short heads of the biceps femoris. Additionally, three muscle tendons were connected to the gluteus maximus. To the best of our knowledge, this is the first report on the third head of the biceps femoris demonstrating two origins, i.e., the proximal part of the femur and the insertion tendon of the gluteus maximus. Moreover, the third head, as well as the short head, of the biceps femoris was innervated by the muscular branch of the common peroneal nerve. Based on the origin and innervation, it can be believed that the third head of the biceps femoris is analogous to its short head and is related to the tenuissimus, a phylogenetic remnant. Therefore, we concluded that this third head is an intermediate muscle type of the tenuissimus and short head of the biceps femoris.

Cardioprotection via Metabolism for Rat Heart Preservation Using the High-Pressure Gaseous Mixture of Carbon Monoxide and Oxygen.

The high-pressure gas (HPG) method with carbon monoxide (CO) and oxygen (O2) mixture maintains the preserved rat heart function. The metabolites of rat hearts preserved using the HPG method (HPG group) and cold storage (CS) method (CS group) by immersion in a stock solution for 24 h were assessed to confirm CO and O2 effects. Lactic acid was significantly lower and citric acid was significantly higher in the HPG group than in the CS group. Moreover, adenosine triphosphate (ATP) levels as well as some pentose phosphate pathway (PPP) metabolites and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were significantly higher in the HPG group than in the CS group. Additionally, reduced glutathione (GSH), which protects cells from oxidative stress, was also significantly higher in the HPG group than in the CS group. These results indicated that each gas, CO and O2, induced the shift from anaerobic to aerobic metabolism, maintaining the energy of ischemic preserved organs, shifting the glucose utilization from glycolysis toward PPP, and reducing oxidative stress. Both CO and O2 in the HPG method have important effects on the ATP supply and decrease oxidative stress for preventing ischemic injury. The HPG method may be useful for clinical application.

Fruit Fly, Drosophila melanogaster, as an In Vivo Tool to Study the Biological Effects of Proton Irradiation.

The clinical superiority of proton therapy over photon therapy has recently gained recognition; however, the biological effects of proton therapy remain poorly understood. The lack of in vivo evidence is especially important. Therefore, the goal of this study was to validate the usefulness of Drosophila melanogaster as an alternative tool in proton radiobiology. To determine whether the comparative biological effects of protons and X rays are detectable in Drosophila, we assessed their influence on survival and mRNA expression. Postirradiation observation revealed that protons inhibited their development and reduced the overall survival rates more effectively than X rays. The relative biological effectiveness of the proton beams compared to the X rays estimated from the 50% lethal doses was 1.31. At 2 or 24 h postirradiation, mRNA expression analysis demonstrated that the expression patterns of several genes (such as DNA-repair-, apoptosis- and angiogenesis-related genes) followed different time courses depending on radiation type. Moreover, our trials suggested that the knockdown of individual genes by the GAL4/UAS system changes the radiosensitivity in a radiation type-specific manner. We confirmed this Drosophila model to be considerably useful to evaluate the findings from in vitro studies in an in vivo system. Furthermore, this model has a potential to elucidate more complex biological mechanisms underlying proton irradiation.