The Origin and Course of the Blood Supply to the Lower Eyelid Orbital Fat: Anatomical Study and Clinical Significance.

PURPOSE: The aim of this study is to investigate the origin and course of the orbital fat arterial supply in the lower eyelid using traditional anatomy and three-dimensional computed tomography (CT). METHODS: Twenty-seven cadaver heads were infused with mercury sulfide contrast media through the ophthalmic artery, maxillary artery, transverse facial artery, and facial artery. CT images were obtained after contrast agent injection, three-dimensional CT scans were reconstructed, and the cadaver heads were dissected. RESULTS: Forty-five qualified hemifaces showed that the orbital fat arterial supply in the lower eyelid originates primarily from the inferomedial muscular trunk (IMT) of the ophthalmic artery and the orbital branch of the infraorbital artery. The medial branch of the IMT terminated at the medial fat pad (35.6%) or the orbital floor (64.4%). The lateral branch terminated at the inferior oblique (IO) muscle (28.9%) or the central and lateral fat pads (17.8%). In 53.3%, the lateral branch extended to the anterior part of the lateral fat pad and terminated in the orbital wall or the zygomaticoorbital foramina. The orbital branch of the infraorbital artery coursed between the orbital floor and the orbital fat, providing supply to the IO muscle, inferior rectus (IR) muscle, nasolacrimal duct, and orbital fat. CONCLUSION: This study elucidated the origin and course of the orbital fat arterial supply in the lower eyelid, which may help to avoid reducing the blood supply of the orbital fat pedicles during surgery. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Inhibition of inosine metabolism of the gut microbiota decreases testosterone secretion in the testis.

Growing evidence indicates that gut microbiota is involved in the regulation of the host's sex hormone levels, such as through interfering with the sex hormone metabolism in the intestine. However, if gut microbiota or its metabolites directly influence the sex hormone biosynthesis in the gonad remains largely unknown. Our previous study showed that colistin, as a narrow-spectrum antibiotic, can significantly downregulate the serum testosterone levels and thus enhance the antitumor efficiency of anti-PD-L1 in male mice; however, the underlying mechanism for the regulation of the host's testosterone levels remains uninvestigated. In the present study, we analyzed the impact of colistin on the immune microenvironment of the testis as well as the composition and metabolism of gut microbiota in male mice. Our results showed that colistin has an impact on the immune microenvironment of the testis and can downregulate serum testosterone levels in male mice through inhibition of Akkermansia, leading to destroyed inosine metabolism. Supplement with inosine can restore testosterone secretion probably by prompting the recovery of the intestinal mucus barrier and the serum lipopolysaccharides levels. All these findings reveal a new pathway for the regulation of the host's sex hormone levels by gut microbiota.IMPORTANCEThis study demonstrates that exposure to even narrow-spectrum antibiotics may affect the host's testosterone levels by altering the gut microbiota and its metabolites. Our findings provide evidence that some specific gut bacteria have an impact on the sex hormone biosynthesis in the testis.

GHS-R1a activity suppresses synaptic function of primary cultured hippocampal neurons.

Ghrelin is an orexigenic gastric hormone that promotes feeding behaviors and regulates energy homeostasis in both humans and rodents. Studies have reported intriguing yet conflicting roles that ghrelin and its receptor growth hormone secretagogue receptor 1a (GHS-R1a) play in regulating multiple brain functions, such as learning and memory; however, the underlying mechanism is largely unknown. In this study, we investigated the effect of ghrelin incubation and virus-mediated GHS-R1a overexpression on synaptic functions of primary cultured hippocampal neurons. Our results demonstrated that ghrelin pre-treatment for 24 h, with a concentration of either 4 nM or 200 nM, suppressed the frequency of miniature excitatory postsynaptic currents (mEPSCs), the frequency and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Similarly, GHS-R1a overexpression inhibited both the frequency and the amplitude of mEPSCs, and mIPSCs frequency. Moreover, our in vitro Ca2+-image study with Rhod-3AM reveals that ghrelin pre-treatment for either 3 h or 24 h suppressed glutamate-induced elevation of cytoplasmic [Ca2+]. Our findings thus suggest that GHS-R1a signaling inhibits synaptic function of hippocampal neurons, which may contribute to the blocking effect of ghrelin on memory formation.

The significance of the neuregulin-1/ErbB signaling pathway and its effect on Sox10 expression in the development of terminally differentiated Schwann cells in vitro.

PURPOSE: The purpose of this study was to explore the significance of the neuregulin-1/ErbB signaling pathway and its effect on Sox10 expression in the course of the differentiation of mouse bone marrow mesenchymal stem cells into Schwann-like cells in vitro. MATERIALS AND METHODS: The experiment was conducted with three groups-control, TAK 165, and HRG-off. In the control group, we used the classical induction method of adding ß-ME, RA, FSK, b-FGF, PDGF, and neuregulin (HRG); the cells were collected on the 7th day. Using the same basic protocol as the control group, the specific ErbB2 inhibitor mubritinib (TAK 165) was added to block the neuregulin-1/ErbB pathway in the TAK 165 group, while HRG was not added in the HRG-off group. We detected the degree of differentiation of stem cells into Schwann-like cells by using RT-PCR to examine the expression of Sox10, NRG-1, ErbB2, ErbB3, and ErbB4 and by using immunofluorescence staining to examine the Schwann cell marker S100B, Glial Fibrillary Acidic Protein (GFAP) and P75. RESULTS: Our results showed that the proliferation of Schwann cells was reduced and apoptosis was increased in the TAK 165 group and the HRG-off group. Sox10 was stably expressed and NRG-1, ErbB2, and ErbB3 increased in the control group. However, the expression of Sox10 in the TAK 165 group was obviously decreased at the end of induced differentiation; meanwhile, the degree of stem cell differentiation also decreased. CONCLUSIONS: the neuregulin-1/ErbB signaling pathway plays an important role in the differentiation of bone marrow mesenchymal stem cells into Schwann-like cells and can promote the maintenance of Sox10 。.

miR-449a Is Related to Short-Term Recurrence of Hepatocellular Carcinoma and Inhibits Migration and Invasion by Targeting Notch1.

PURPOSE: To explore the effect of miR-449a inhibits migration and invasion by targeting Notch1 and regulating epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC), and further study on the molecular mechanism. PATIENTS AND METHODS: The expression of miR-449a and Notch1 in HCC cells and tissues was detected by qRT-PCR. The HCC cell line HCCLM3 and SMMC-7721 were transfected with miR-449a. The invasion and migration of HCC cell lines were detected by transwell assay and wound healing assay. The Notch pathway and EMT related protein were detected with Western Blotting. The specific binding site of mir-449a on notch1 gene was detected by luciferase assay. RESULTS: We found the expression of miR-449a was related to short-term recurrence of hepatocellular carcinoma after hepatectomy and acted as independent risk factors of DFS and OS. The expression of miR-449a decreased in tumor tissues and HCC cell lines, but the expression of Notch1 increased. The overexpressed miR-449a promoted the invasiveness in vitro by regulating EMT via Notch pathway. Mechanically, miR-449a inhibited the translation of Notch1 protein by binding to 3' UTR of its mRNA directly. CONCLUSION: miR-449a is short-term recurrence-related miRNA and inhibits the invasion and metastasis ability of HCC cells by regulating EMT via Notch pathway. miR-449a may be a new effective therapeutic target for HCC.

NEDD9 promotes invasion and migration of colorectal cancer cell line HCT116 via JNK/EMT.

Neural precursor cell-expressed, developmentally-downregulated 9 (NEDD9) is a multi-domain skeleton protein that serves an important role in the cell signaling process via modulating invasion, metastasis, proliferation and apoptosis of tumor cells. The present study identified that the expression levels of NEDD9 in colorectal cancer were elevated. Therefore, the effect of downregulating the expression of NEDD9 in terms of invasion and migration of colorectal cancer cells was investigated and the role of the JNK pathway in these processes was also investigated. The data revealed that downregulation of NEDD9 and JNK inhibitors suppressed invasion and migration, decreased expression levels of phosphorylated JNK, increased the expression levels of E-cadherin and decreased the expression levels of vimentin. In summary, NEDD9 promotes invasion and migration of colorectal cancer cells via the JNK pathway.

MiR-449a regulates the cell migration and invasion of human non-small cell lung carcinoma by targeting ADAM10.

Background: MicroRNAs (miRNAs) are non-coding small RNAs that have been shown to play a key role in the development of many tumors. However, its specific mechanism of action in non-small cell lung cancer (NSCLC) is not very clear. Purpose: This study was to identify the effect of miRNA-449a on NSCLC invasion and migration. Methods: We used quantitative real-time PCR experiments to demonstrate that miRNA-449a is down-regulated in NSCLC tissues and cell lines. We also used the Transwell assay to detect cell invasion and migration, and the Western Blot assay  was used to detect protein expression. The dual luciferase assay was used to detect the targeting relationship between miR-449a and A Disintegrin And Metalloproteinases 10 (ADAM10). Results: Our experiments demonstrated that miRNA-449a was down-regulated in NSCLC tissues and cell lines. When miRNA-449a was up-regulated in NSCLC cells, the invasion and migration ability of the cells was weakened, and the expression of ADAM10 was decreased. After down-regulation of miRNA-449a, the cell's invasion and migration ability was enhanced, and the expression of ADAM10 was increased. Through dual luciferase assays, we also found that miRNA-449a can target ADAM10 to delay the progression of epithelial-mesenchymal transition (EMT) and inhibit invasion and migration. Conclusion: Our experiments demonstrated that miRNA-449a acted as a tumor suppressor gene through inhibiting the expression of ADAM10 in NSCLC.

Th17 cells regulate the production of CXCL1 in breast cancer.

Recently, the link between inflammation and cancer has been targeted for the prevention or treatment of malignant tumours. We aimed to investigate the relationship between Th17 cells and CXCL1 in breast cancer and the biological effects of CXCL1 on breast cancer. In vivo, the Th17 cell frequency in the peripheral blood was determined by flow cytometry. Secretion of IL-17 and CXCL1 in the blood serum was determined by enzyme-linked immunosorbent assay (ELISA). Expression of IL-17A and CXCL1 mRNA was determined by qRT-PCR. In vitro, the effects of Th17/CXCL1 during breast cancer were assessed in the human breast cancer cell lines MCF-7 and MDA-MB-231. Cell proliferation was measured using the CCK8 assay. Cell invasion and migration ability were assessed using a transwell cell invasion and wound- healing assay. In vivo, Th17 cells and CXCL1 were increased in breast cancer patients. Moreover, their changes were correlated in breast cancer cells. Th17 cells upregulate the production of CXCL1 during breast cancer progression. CXCL1, which is produced by breast cancer cells, can promote cancer growth and development, and may also point to a specific histogenetic pathway.

Reduced inflammatory factor expression facilitates recovery after sciatic nerve injury in TLR4 mutant mice.

Toll-like receptors (TLRs) are extremely significant pattern recognition receptors. When nerve injury occurs, a variety of inflammatory factors are generated, leading to an exceedingly complex micro-environment. TLRs recognize damage-associated molecular patterns. To investigate the correlation between TLR4 and recovery after sciatic nerve injury, the model of sciatic nerve injury was conducted using TLR4-mutated mice (C3H/HeJ) and wild mice (C3H/HeN). Our goal was to identify short-stage and long-stage changes after sciatic nerve injury, mainly by checking the expression changes of inflammation factors in the short-stage and the differences in the recovery of the injured sciatic nerve in the long-stage. The results show that the increase of changes in the HeN group of IL-1ß, IL-6, TNF-α and MCP-1 are more obvious than in the HeJ group, with caspase1 expression higher and Nlrp3 expression lower in the former group. Further results reveal intense inflammation occurred in the HeN group showing more neutrophils and macrophages. Nlrp3 and caspase1 showed little difference by Immunohistochemistry, with Nlrp6 expression differing between the HeJ group and the HeN group. The results led us to conclude that better recovery of the injured sciatic nerve occurred in the HeJ group because the expression of GAP-43 and p75NTR was higher and had a better SFI figure. TLR4 mutation can decrease the expression of inflammatory factors and enhance the speed of recovery after sciatic nerve injury. The changes in the expression of Nlrp6, which are related to the TLR4 mutation, may influence recovery of the injured sciatic nerve. Further studies will be conducted to confirm these results.

Silencing of Prrx2 Inhibits the Invasion and Metastasis of Breast Cancer both In Vitro and In Vivo by Reversing Epithelial-Mesenchymal Transition.

BACKGROUND/AIMS: Epithelial-mesenchymal transition (EMT) is recognized as a crucial mechanism in breast cancer progression and metastasis. Paired-related homeobox 2 (Prrx2) has been identified as a new EMT inducer in cancer, but the underlying mechanisms are still poorly understood. METHODS: The expression of Prrx2 was assessed by immunohistochemistry in breast cancer tissues to evaluate the clinicopathological significance of Prrx2, as well as the correlation between Prrx2 and EMT. Short hairpin RNA knockdown of Prrx2 was used to examine cellular effects of Prrx2, detecte the expression of Wnt/ß-catenin signaling and EMT-associated proteins, and observe cell proliferation, invasion and migration abilities in vitro and in vivo. RESULTS: Clinical association studies showed that Prrx2 expression was related to tumor size, lymph node metastasis, tumor node metastasis stages, EMT and poor survival. Results also showed that knockdown of Prrx2 could alter cell morphology, suppressed the abilities of cell proliferation, invasion and migration in breast cancer. Moreover, silencing of Prrx2 induced the mesenchymal-epithelial transition and prevented nuclear translocation of ß-catenin, inhibited wnt/ß-catenin signaling pathway. CONCLUSION: Our study indicated that Prrx2 may be an important activator of EMT in human breast cancer and it can serve as a molecular target of therapeutic interventions for breast cancer.

Notch signaling mediated by TGF-ß/Smad pathway in concanavalin A-induced liver fibrosis in rats.

AIM: To explore the exact interaction between Notch and transforming growth factor (TGF)-ß signaling in liver fibrosis. METHODS: We established a rat model of liver fibrosis induced by concanavalin A. Peripheral blood mononuclear cells (PBMCs) were isolated from the modeled rats, and cultured with γ-secretase inhibitor DAPT and TGF-ß inhibitor for 24 h. The mRNA levels of Notch and TGF-ß signaling were detected by quantitative real-time polymerase chain reaction. Expression of Notch and TGF-ß proteins was analyzed by western blotting. RESULTS: Compared to control rats, Notch and TGF-ß signaling was activated in PBMCs of model rats. Administration of DAPT and TGF-ß inhibitor suppressed Notch and TGF-ß signal transducer in PBMCs of model rats. DAPT reduced mRNA and protein expression of TGF-ß signaling, such as TGF-ß1 and Smad3. TGF-ß inhibitor also downregulated Notch1, Hes1 and Hes5, and mRNA and protein expression of the Notch signaling pathway. CONCLUSION: Notch and TGF-ß signaling play a role in liver fibrosis. TGF-ß signaling upregulates Notch signaling, which promotes TGF-ß signaling.

Lack of an association between XRCC2 R188H polymorphisms and breast cancer: an update meta-analysis involving 35,422 subjects.

PURPOSE: Several studies have investigated the associations between XRCC2 R188H polymorphism and the susceptibility to breast cancer, but the results have been inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. METHODS: PubMed and China National Knowledge Infrastructure (CNKI) searches were carried out for relevant studies published before March 2015. Meta-analysis was performed with the Stata, version 11.0. RESULTS: A total of 17 case-control studies, including 17,986 cases and 17,436 controls, were selected. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association in the homozygous model, dominant model, and recessive model. When all the studies were pooled into the meta-analysis, there was no evidence showing a significant association between XRCC2 R188H polymorphism and breast cancer risk (for homozygous model, OR=0.84, 95% CI=0.62-1.14; for dominant model: OR=0.76, 95% CI=0.53-1.09; and for recessive model: OR=1.04, 95% CI=0.98-1.10). In the subgroup analysis by ethnicity, no significant association was found between the polymorphism and breast cancer risk. CONCLUSIONS: In conclusion, this meta-analysis indicates that the XRCC2 R188H polymorphism is not a risk factor for developing of breast cancer.

Regional tissue immune responses after sciatic nerve injury in rats.

Inflammatory cells play a critical role during nerve regeneration following peripheral nerve injury. In this study, we investigated immune responses in rat sciatic nerve after injury. Wistar rats were randomly divided into the sciatic nerve injury (model) group and control group. The right sciatic nerve of rats in the model group was transected and sutured end-to-end. Our results showed that rats in the model group functionally recovered following sciatic nerve injury. We detected inflammatory cell infiltration in the remaining sciatic nerves following injury. In addition, expression of interferon-γ (INF-γ), interleukin-10 (IL-10), and the INF-γ/IL-10 ratio was significantly elevated one week following nerve injury, but gradually decreased thereafter. Our findings demonstrate that immune responses and inflammatory cell activation are involved during recovery from sciatic nerve injury.

[Expression Changes of Matrix Reconstitution-related Genes in Bone Marrow during 5-Fluorouracil-Induced Hemopoietic Injury and Their Significance].

OBJECTIVE: To explore the expression change of the genes related with matrix reconstitution during the injury and reconstitution of murine bone marrow following treatment with 5-fluorouracil (5-FU). METHODS: C57BL/6 mice received intraperitoneal injection of 5-FU (200 mg/kg), and peripheral blood cell counts were monitored at 3, 6, 9, 15, 21, 27 days after treatment. Bone marrow cells were harvested at these times for total RNA extraction using TRIzol. Reverse transcriptions in combination with real-time PCR were performed for detecting expression of genes related with matrix reconstitution, including ECM-1, MMP-2, MMP-3, MMP-13 and TIMP-1. RESULTS: After injection of 5-FU, the numbers of three line cells in peripheral blood (i.e. RBC, WBC and platelets) decreased and then recovered with differential dynamics. Similarly, RT-qPCR revealed that all the 5 detected gene expressions were significantly up-regulated during the injury. The mRNA expression of MMP-2 reached to peak at day 3 while the other genes reached to peak at day 6. MMP-3 has a low expression when compared with others, but its expression increased significantly after injury. CONCLUSION: In 5-FU induced hematopoietic injury and reconstitution model, matrix reconstitution-related genes may play an important role in hematopoietic reconstitution, but different genes play different roles at various time, and cooperate with each other for hematopoietic reconstitution.

Preventive effect of halofuginone on concanavalin A-induced liver fibrosis.

Halofuginone (HF) is an active component of extracts derived from the plant alkaloid febrifugine and has shown therapeutic promise in animal models of fibrotic disease. Our main objectives were to clarify the suppressive effect of HF on concanavalin A (ConA)-induced liver fibrosis. ConA injection into the tail vein caused a great increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, while orally administration of HF significantly decreased the levels of the transaminases. In addition, the levels of hyaluronic acid (HA), procollagen III (PCIII) and TGF-ß1 in the serum and collagen I, α-SMA, tissue inhibitors of metalloproteinase 2 (TIMP2) and Smad3 in the liver tissue were significantly lowered with the treatment of HF. Histological examination also demonstrated that HF significantly reduced the severity of liver fibrosis. Since ConA-induced liver fibrosis is caused by the repeated activation of T cells, immunomodulatory substances might be responsible for the suppressive effect of HF. We found that the production of nuclear factor (NF)-kB in the serum was increased in ConA-treated group, while decreased significantly with the treatment of HF. The changes of inflammatory cytokines tumor necrosis factor (TNF-α), IL-6 and IL-1ß in the serum followed the same rhythm. All together, our findings indicate that orally administration HF (10ppm) would attenuate the liver fibrosis by suppressing the synthesis of collagen I and inflammation-mediated liver injury.

Preparation and influencing factors of sirolimus liposome by supercritical fluid.

We prepared the sirolimus liposome by rapid expansion of supercritical solution (RESS) technology of supercritical fluid, and studied the effects of temperature, pressure, and equilibrium time on the average particle size and envelop rate of liposome. The conditions of the minimal average particle size of liposome were 328K of temperature (35MPa), 35MPa of pressure (333K), and 50 minutes of equilibrium (343K) time, respectively. The conditions of the maximal envelop rate of liposome were 333K of temperature (30MPa), 35MPa of pressure (343K), and 50 minutes of equilibrium (323K) time, respectively.

A neuropathological study at autopsy of early onset spinocerebellar ataxia 6.

Spinocerebellar ataxia type 6 (SCA6) is a late-onset, autosomal dominantly inherited ataxic disorder, and most previous clinical studies consider SCA6 to be a "pure" cerebellar ataxia. We carried out a detailed pathoanatomical study at autopsy of two patients, brother and sister, with genetically confirmed SCA6. The disease in both patients was early onset and short, which is atypical for SCA6. We observed severe neurodegeneration in the cerebellum, dentate nucleus and olivary nuclei. Both patients showed evidence of synaptic modification in the cerebellar cortex, which morphologically confirmed the existence of retrograde and anterograde trans-synaptic degeneration secondary to the cerebellar cortical lesion. Furthermore, our study shows for the first time that neurodegeneration in SCA6 occurs in the spinal cord. Finally, our postmortem study confirms that SCA6 is not a simple "pure" cerebellar disease, but a complex neurodegenerative condition in which many extracerebellar structures are involved.

Spinocerebellar ataxia type 6: Systematic patho-anatomical study reveals different phylogenetically defined regions of the cerebellum and neural pathways undergo different evolutions of the degenerative process.

Spinocerebellar ataxia type 6 is a late onset autosomal dominantly inherited ataxic disorder, and previous patho-anatomical studies have only reported neurodegeneration in SCA6 as being confined to the cerebellar cortex, dentate nucleus and inferior olive. However, the characteristics of cerebellar symptoms and many poorly understood "extracerebellar" symptoms reveal the three cerebellar regions and the corresponding precerebellar nuclei may undergo differing evolution of the degenerative process, and a more widespread brainstem degeneration in SCA6. We carried out a detailed immunohistochemical study in two SCA6 patients who had rather early onset and short disease duration with 25 CAG repeats, which is atypical for SCA-6. We investigated the severity of neurodegeneration in each of the cerebellar regions and the corresponding precerebellar nuclei, and further characterize the extent of brain degeneration. This study confirmed that vestibulocerebellar, spinocerebellum and pontocerebellar are consistent targets of the pathological process of SCA6, but the severity of neurodegeneration in each of them was different. Vestibulocerebellum and the inferior cerebellar peduncle undergo the most severe neurodegeneration, while neurodegeneration in the pontocerebellar is less severe. Furthermore, we observed obvious neurodegeneration in layers II and III of the primary motor cortex, vestibular nuclei, inferior olivary nucleus, nucleus proprius and posterior spinocerebellar tract. Our detailed postmortem findings confirmed that SCA6 was not a simple "pure" cerebellar disease, but a complex neurodegenerative disease in which the three cerebellar regions underwent different evolutions of neurodegeneration process, and the corresponding precerebellar nuclei and the neural pathway were all involved.

Relationship between CX3CR1 genetic polymorphism and carotid atherosclerosis.

To investigate the possible association between V249I and T280M polymorphisms and carotid atherosclerosis (CAS) in a Han population in northern China we studied 328 patients with increased carotid artery intima-media thickness (C-IMT) and 292 healthy controls with normal C-IMT. T280M and V249I polymorphic genotypes of CX3CR1 were determined by polymerase chain reaction restriction fragment length polymorphism. Results showed that the M280 allele frequency in CAS group was significantly lower than that in the controls. Logistic regression analysis revealed that the decreased frequency of the M280 allele was an independent risk factor for CAS.