Myeloid-derived suppressor cells in head and neck squamous cell carcinoma.

Myeloid-derived suppressor cells (MDSCs), which originated from hematopoietic stem cells, are heterogeneous population of cells that have different differentiation patterns and widely presented in tumor microenvironment. For tumor research, myeloid suppressor cells have received extensive attention since their discovery due to their specific immunosuppressive properties, and the mechanisms of immunosuppression and therapeutic approaches for MDSCs have been investigated in a variety of different types of malignancies. To improve the efficacy of treatment for head and neck squamous cell carcinoma (HNSCC), a disease with a high occurrence, immunotherapy has gradually emerged in after traditional surgery and subsequent radiotherapy and chemotherapy, and has made some progress. In this review, we introduced the mechanisms on the development, differentiation, and elimination of MDSCs and provided a detailed overview of the mechanisms behind the immunosuppressive properties of MDSCs. We summarized the recent researches on MDSCs in HNSCC, especially for targeting-MDSCs therapy and combination with other types of therapy such as immune checkpoint blockade (ICB). Furthermore, we looked at drug delivery patterns and collected the current diverse drug delivery systems for the improvement that contributed to therapy against MDSCs in HNSCC. Most importantly, we made possible outlooks for the future research priorities, which provide a basis for further study on the clinical significance and therapeutic value of MDSCs in HNSCC.

Long Non-Coding RNA (lncRNA) 91H Confers Tamoxifen Resistance in ER+ Breast Cancer Cells through Inhibiting mTOR Signaling Pathway.

OBJECTIVE: Estrogen receptor-positive (ER+) breast cancers are the most often diagnosed subtype of breast tumors, in which the development of tamoxifen resistance remains a major impediment. The effect of long non-coding RNA (lncRNA) on therapy resistance is beginning to emerge. The lncRNA 91H, a recently identified lncRNA involved in tumorigenesis, is also overexpressed in breast cancer. The purpose of this study was to explore the role of 91H in the biological function and tamoxifen resistance of ER+ breast cancer cells. METHODS: MCF-7 and T47D cells were transfected for 91H silence. CCK-8 assay was performed to examine cell viability and drug sensitivity. Cell cycle and apoptosis were analyzed using flow cytometry. Cell migration capacity was determined by wound healing assay. The protein level was analyzed by Western blotting. RESULTS: MCF-7 and T47D cells with 91H knockdown exhibited lower capacities of cell proliferation and migration. In addition, knockdown of 91H resulted in significantly increased sensitivity to tamoxifen and a higher ratio of apoptosis induced by tamoxifen. Furthermore, the protein level of p-mTOR was notably inhibited through downregulating 91H expression. And the mTOR inhibitor together with tamoxifen presented synergistic effect on the inhibition of cell viability. CONCLUSION: Our study highlights that 91H might serve as a potential target for ER+ breast cancer patients who have acquired tamoxifen resistance.

Long Non-coding RNA LINC02195 as a Regulator of MHC I Molecules and Favorable Prognostic Marker for Head and Neck Squamous Cell Carcinoma.

The loss of major histocompatibility complex class I (MHC I) molecules is an important mechanism by which cancer cells escape immunosurveillance in head and neck squamous cell carcinoma (HNSCC). Several long non-coding RNAs (lncRNAs) have been implicated in immune response and regulation including antigen processing and presentation. However, few studies on lncRNAs regulating MHC I expression in HNSCC have been conducted. In this study, MHC I related lncRNAs were identified from the The Cancer Genome Atlas (TCGA) HNSCC database. One of the lncRNAs, long intergenic non-protein coding RNA 2195 (LINC02195), was found to be associated with genes encoding MHC I molecules and patient prognosis in the TCGA database. KEGG and GO analyses suggested that LINC02195 was closely related to antigen processing and presentation. qRT-PCR revealed high expression of LINC02195 in human HNSCC tissues and HNSCC cell lines compared with normal mucosal tissues. in situ hybridization of the HNSCC tissue microarray revealed a correlation between high LINC02195 expression and a favorable prognosis in our patient cohort. Silencing of LINC02195 decreased MHC I protein expression, as evidenced by western blotting. Multiplex immunochemistry was performed to reveal the positive correlation between high LINC02195 expression and an increased number of CD8+ and CD4+ T cells in the tumor microenvironment. Based on our study, LINC02195 is a promising prognostic marker and a target for future therapeutic interventions.

Identification of an lncRNA­miRNA­mRNA interaction mechanism in breast cancer based on bioinformatic analysis.

Non­coding RNAs serve important roles in regulating the expression of certain genes and are involved in the principal biological processes of breast cancer. The majority of studies have focused on defining the regulatory functions of long non­coding RNAs (lncRNAs) and microRNAs (miRNAs/miRs), and few studies have investigated how lncRNAs and miRNAs are transcriptionally regulated. In the present study, based on the breast invasive carcinoma dataset from The Cancer Genome Atlas at cBioPortal, and using a bioinformatics computational approach, an lncRNA­miRNA­mRNA network was constructed. The network consisted of 601 nodes and 706 edges, which represented the complex web of regulatory effects between lncRNAs, miRNAs and target genes. The results of the present study demonstrated that miR­510 was the most potent miRNA controller and regulator of numerous target genes. In addition, it was observed that the lncRNAs PVT1, CCAT1 and linc00861 exhibited possible interactions with clinical biomarkers, including receptor tyrosine­protein kinase erbB­2, estrogen receptor and progesterone receptor, demonstrated using RNA­protein interaction prediction software. The network of lncRNA­miRNA­mRNA interactions will facilitate further experimental studies and may be used to refine biomarker predictions for developing novel therapeutic approaches in breast cancer.

A 3D navigation template for guiding a unilateral lumbar pedicle screw with contralateral translaminar facet screw fixation: a study protocol for multicentre randomised controlled trials.

INTRODUCTION: The incidence of lumbar disc degeneration disease has increased in recent years. Lumbar interbody fusion using two unilateral pedicle screws and a translaminar facet screw fixation has advantages of minimal invasiveness and lower costs compared with the traditional methods. Moreover, a method guided by a three-dimensional (3D) navigation template may help us improve the surgical accuracy and the success rate. This is the first randomised study using a 3D navigation template to guide a unilateral lumbar pedicle screw with contralateral translaminar facet screw fixation. METHODS AND ANALYSIS: Patients who meet the criteria of the surgery will be randomly divided into experimental groups and control groups by a computer-generated randomisation schedule. We will preoperatively design an individual 3D navigation template using CATIA software and MeditoolCreate. The following primary outcomes will be collected: screw angles compared with the optimal screw trajectories in 3D digital images, length of the wound incision, operative time, intraoperative blood loss and complications. The following secondary outcomes will be collected: visual analogue scale (VAS) for back pain, VAS for leg pain and the Oswestry Disability Index. These parameters will be evaluated on day 1 and then 3, 6, 12 and 24 months postoperatively. ETHICS AND DISSEMINATION: The study has been reviewed and approved by the institutional ethics review board of the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University. The results will be presented at scientific communities and peer-reviewed journals. TRIAL REGISTRATION NUMBER: ChiCTR-IDR-17010466.

[Unilateral pedicle screw fixation combined with contralateral percutaneous translaminar facet screw fixation and lumbar interbody fusion for the treatment of lower lumbar diseases: an analysis of complications].

OBJECTIVE: To investigate the features and causes of complications of unilateral pedicle screw fixation combined with contralateral percutaneous translaminar facet screw fixation and lumbar interbody fusion in treating lower lumbar diseases. METHODS: The clinical data of 166 patients with lower lumbar diseases who underwent unilateral pedicle screw fixation combined with contralateral percutaneous translaminar facet screw fixation and lumbar interbody fusion with intervertebral cages from January 2008 to December 2013 were retrospectively analyzed. There were 64 males and 102 females, aged from 24 to 74 years with a mean of 51.9 years old, suffered from lower lumbar lesions for 47.5 months on average (ranged, 8 months to 30 years). Among these patients, lumbar intervertebral disc degeneration was found in 49 patients, recurred lumbar intervertebral disc protrusion in 17 patients, massive lumbar intervertebral disc protrusion in 23 patients, lumbar intervertebral disc protrusion accompany with spinal canal stenosis in 27 patients, lumbar degenerative spondylolisthesis with degree I (Meyerding grade) in 21 patients, far lateral lumbar intervertebral disc protrusion in 5 patients. Single segmental diseases occurred in 124 patients and two segmental diseases in 42 patients. The diseases occurred at L(3,4) segment in 6 patients, at L(4,5) segment in 97 patients, at L5S1 segment in 21 patients, at L(2,3), and L(3,4) segments in 1 patient, at L(3,4) and L4,5) segments in 26 patients, and at L(4,5), and L5S1 segments in 15 patients. RESULTS: There was no abnormal bleeding in the patients and no patient received blood transfusion. During the surgery, spinal dura mater injury with cerebrospinal fluid leakage complicated in 1 patient, a fracture of vertebral pedicle in 4 patients, and end plate injury in 2 patients. No postoperative cerebrospinal fluid, incision infection and skin necrosis were found after operation. Nerve root injury was found in 1 patient. According to the position of pedicles crew, 371 screws of 163 patients were in degree I and 3 screws of 3 patients were in degree II; position of translaminar facet screw, 199 screws of 157 patients were type I, 8 screws of 8 patients were type II, 1 screw of 1 patient was III. Translaminar facet screw was slightly short in 2 patients. Five patients were lost to follow-up, two patients were died. The remaining patients were followed up for 35.4 months on average (ranged, 12 to 60 months). During the follow-up period , end plate was cut off and intervertebral cages were embedded in 14 segments of 14 patients. Abnormal pain of both lower extremities was found in 1 patient. With the exception of 11 unidentified segments in 11 patients, 189 segments of 148 patients obtained intervertebral fusion. No loosening, displacement, breakage of pedicle screw or translaminar facet screw, displacement of intervertebral cages or obvious degeneration of adjacent segments were found. The coronal and sagittal planes balance of lumbar vertebra were obviously improved. Postoperative JOA score was significantly increased than that of preoperative. CONCLUSION: Unilateral pedicle screw fixation combined with contralateral percutaneous translaminar facet screw fixation and lumbar interbody fusion with intervertebral cages is a good choice for the treatment of lower lumbar diseases, but it has a risk of complications. Abundant surgeon's surgical experience, careful operation, and rational use of imaging technique can effectively reduce the incidence of complications.

[Unilateral pedicle screw fixation versus its combination with contralateral translaminar facet screw fixation for the treatment of single segmental lower lumbar vertebra diseases].

OBJECTIVE: To compare the advantages and disadvantages of unilateral pedicle screw fixation plus lumbar interbody fusion and unilateral pedicle screw fixation combined with contralateral translaminar facet screw fixation plus lumbar interbody fusion in treating single segmental lower lumbar vertebra diseases. METHODS: Sixty-two patients with single segmental lower lumbar vertebra disease who received treatment between January 2008 and June 2009. These patients were consisted of 16 males and 46 females, ranging in age from 27 to 72 years old, with a mean age of 51.6 years old. Among these patients, lumbar degenerative disease had in 22 patients, recurrence of lumbar intervertebral disc protrusion in 13 patients, lumbar intervertebral disc protrusion accompany with spinal canal stenosis in 12 patients, massive lumbar intervertebral disc protrusion in 5 patients and lumbar degenerative spondylolisthesis with degree I in 10 patients. The lesions occurred at L3,4 segment in 5 patients, at L4,5 segment in 42 patients, and at L5S1 segment in 15 patients. Thirty patients underwent unilateral pedicle screw fixation (unilateral screw fixation group, group A) and thirty-two patients received unilateral pedicle screw fixation combined with contralateral translaminar facet screw fixation (bilateral screw fixation group, group B). Lumbar interbody fusion with intervertebral cages was also performed in all patients. Incision length, operation time, intraoperative blood loss and postoperative wound drainage were compared between two groups. Loosening or breakage of internal fixations, displacement of intervertebral cages and interbody fusion conditions were observed in each group. Preoperative and postoperative intervertebral height, coronal and sagittal Cobb angle and wound pain at 72 h after operation were compared between two groups. The Japanese Orthopedic Association (JOA) scoring system was used to evaluate the cinical effects. RESULTS: Neither wound infection, skin necrosis, nerve root or cauda equia injury, nor worsened neurological dysfunction in the lower limb occurred in each group. There were no significant differences in incision length, intraoperative blood loss and postoperative wound drainage between two groups. The operation time in group A was significantly shorter than that of group B (P < 0.05). There were no significant differences in visual analogue scale value of the wound pain at postoperative 72 h between two groups (P > 0.05). All patients were followed up for 12-48 months,with a mean of 27.5 months. The intervertebral height of all patients had obviously recovered at 5 days after operation, furthermore, at the final follow-up, it still had well maintained. During follow-up, no pedicle screw and/or translaminar facet screw loosening, displacement or breakage and displacement of intervertebral cages were found. The lumbar interbody fusion rate was 96.7% and 96.9% in group A and group B, respectively, and there was no significant difference between two groups (P > 0.05). JOA score of all patients got obviously improved after operation (P < 0.05) and there was no significant difference between two groups (P > 0.05). CONCLUSION: Both unilateral pedicle screw fixation plus lumbar interbody fusion and unilateral pedicle screw fixation combined with contralateral translaminar facet screw fixation plus lumbar interbody fusion have advantages of small incision, minimal invasion, simple operation, reliable stability, high interbody fusion rate,rapid recovery, encouraging clinical effects and less complications. Compared with unilateral pedicle screw fixation combined with contralateral translaminar facet screw fixation, the operation of unilateral pedicle screw fixation is simpler and can avoid using special equipments. Therefore, unilateral pedicle screw fixation plus lumbar interbody fusion can be used in treating single-segmental lower lumbar vertebra diseases under the precondition of strictly grasping indications for surgery and improving surgical skills.

[Two different fixation methods combined with lumbar interbody fusion for the treatment of two-level lumbar vertebra diseases: a clinical comparison study].

OBJECTIVE: To investigate the advantages and disadvantages of unilateral pedicle screw fixation combined with contralateral translaminar facet screw fixation and interbody fusion with cages in the treatment of two-level lumbar vertebra diseases, by comparing bilateral pedicle screw fixation and interbody fusion with cages. METHODS: Forty-nine patients with two-level lumbar diseases who received treatments from June 2009 to December 2011 were included in this study. Among these patients, 23 patients received unilateral pedicle screw fixation combined with contralateral translaminar facet screw fixation and interbody fusion with cages (combined fixation group) and the remaining 26 patients underwent bilateral pedicle screw fixation and interbody fusion with cages (bilateral fixation group). These patients consisted of 17 males and 32 females, ranging in age from 29 to 68 years old. Among these patients, lumbar intervertebral disc herniation accompanied by the spinal canal stenosis was found in 29 patients, degenerative lumbar disc diseases in 17 patients and lumbar degenerative spondylolisthesis (degree I) in 3 patients. The lesions occurred at L2,3 and L3,4 segments in 1 patient, at L3,4 and L4,5 segments in 30 patients, and at L4,5 segment and L5S1 segment in 18 patients. Wound length, operation time, intraoperative blood loss and postoperative wound drainage were compared between two groups. Intervertebral space height in the lesioned segment before and during surgery and at the latest follow up was also compared between two groups. Before surgery and at the latest follow-up, the Cobb angle of the coronal plane and sagittal plane of the lumbar spine, loosening or breakage of internal fixations, the dislocation of intervertebral cages, and interbody fusion were all evaluated in each group. The visual analogue scale (VAS) was used to measure lumbar incision pain. The Japanese Orthopedic Association (JOA) scoring system was used to evaluate the function before surgery and at the latest follow-up. RESULTS: No wound infection or skin necrosis was observed after surgery in all patients. No cerebrospinal fluid leakage, nerve root injury, cauda equia injury or worsened neural function in the lower limb occurred in all patients during and after surgery. Wound length, operation time, intraoperative blood loss and postoperative wound drainage in the combined fixation group were superior to those in the bilateral fixation group. At postoperative 72 hours, the VAS score in the combined fixation group (1 to 4 points, mean 2.35±1.20) was significantly lower than that in the bilateral fixation group (2 to 5 points, mean 3.11±1.00; P<0.05). All the patients were followed up for 12 to 48 months, with a mean of 29 months. After surgery, intervertebral space height was well recovered in each patient and it was well maintained at the latest follow-up, and there was no significant difference between two groups (P>0.05). During follow-up, pedicle screw and translaminar facet screw loosening, dislocation or breakage and dislocation of intervertebral cages were all not found. At the latest follow-up, the Cobb angle of the coronal plane and sagittal plane of the lumbar spine was obviously improved and was not significantly different between two groups (P>0.05). The lumbar interbody fusion rate was 93.5% and 96.2% in the combined fixation group and bilateral fixation group, respectively, and there was no significant difference between them (P>0.05). There was a significant difference in JOA score between before surgery and at the latest follow-up in each patient (P<0.05), and at the latest follow-up, significant difference in JOA score was found between two groups (P<0.05). CONCLUSION: Compared to bilateral pedicle screw fixation and lumbar interbody fusion with cages, unilateral pedicle screw fixation combined with contralateral translaminar facet screw fixation and lumbar interbody fusion with cages shows advantages including small skin incision, minimal invasion, ease of operation, highly reliable stability, high interbody fusion rate, rapid recovery in the treatment of two-level lumbar vertebra diseases and therefore can be preferred as a treatment method of this disease.

Mixed Fixation and Interbody Fusion for Treatment Single-Segment Lower Lumbar Vertebral Disease: Midterm Follow-up Results.

OBJECTIVE: To investigate the midterm outcomes of unilateral pedicle screws combined with contralateral gunsight-guided translaminar facet screw fixation and interbody fusion for treatment of single-segment lower lumbar vertebral disease. METHODS: A total of 78 patients with various lower lumbar single-segment vertebral diseases were treated in our department from January 2008 to December 2011. There were 21 males and 57 females, with an average age of 52.5 years, including lumbar disc degeneration (28 cases), local recurrence of lumbar disc herniation (9 cases), huge lumbar disc herniation (11 cases), lumbar disc herniation with spinal stenosis (13 cases), and lumbar degenerative spondylolisthesis (degree I) (17 cases). All patients were treated by unilateral pedicle screws in the median incision combined with contralateral gunsight-guided translaminar facet screw fixation and interbody fusion. RESULTS: No wound infection or skin necrosis around incision was observed after operation. No leakage of cerebrospinal fluid or nerve injury occurred during and after operation. Excepting 4 cases, 74 cases were followed up for 18-60 months, averaged 33.5 months. All but one patient (98.6%) received interbody fusion. The intervertebral height of the indexed level was well restored and maintained. At final follow-up. During follow-up, there was no screw loosening or pedicle fracture observed. No apparent degeneration of adjacent segments. The mean Japanese Orthopaedic Association (JOA) scores was increased significantly from 12.79 ± 2.12 preoperatively to 25.8 ± 2.87 at the final follow-up. CONCLUSION: Unilateral pedicle screw fixation combined with contralateral translaminar facet screw fixation and interbody fusion showed good mid-term outcomes in the treatment of single-segment lower lumbar vertebral disease, and can be used as an optimal choice for fixation and fusion of some single-segment lower lumbar vertebral diseases.

[Injury characteristics and surgical treatment of lower lumbar vertebral burst fractures].

OBJECTIVE: To investigate the feasibility of utilizing self-designed score system for lower lumbar vertebral burst fractures to select surgical approach. METHODS: From January 2006 to December 2011, the clinical data of 56 patients with lower lumbar vertebra burst fractures who underwent surgical treatment were retrospectively analyzed. There were 42 males and 14 females with an average age of 43.1 years old (ranged, 19 to 65). Causes of injury included falling down (40 cases), traffic accidents (12 cases), and crashing injury by heavy objects(4 cases). Injury site was L3 in 37 cases, L4 in 16 cases, and L5 in 3 cases. According to the AO classification, 17 cases were type A3.1, 14 cases were type A3.2, 25 cases were type A3.3. According to Frankel grade of nerous function, 2 cases were grade B, 5 cases were grade C, 9 cases were grade D and 40 cases were grade E. Surgical methods and approaches were chosen based on the comprehensive evaluation of AO classification, condition of posterior column injury and spinal canal encroachment. Surgical methods and approaches included trans-vertebra fixation (15 cases), intra-vertebra pedicle screw fixation (21 cases), combination of anterior and posterior approaches (11 cases), one-stage posterior approaches (9 cases). Cobb angles, restorations of the affected vertebral anterior border height, and conditions of spinal canal encroachment were compared before and after surgery. Conditions of bone graft fusion and internal fixation (if bending, loosening or breakage existed) were observed. Spinal cord functions were assessed according to Frankel grade. Localized pain and working status of patients were also assessed at the last follow-up. RESULTS: No incision infection was found and no spinal nerve symptoms improved in all of 56 patients. All patients were followed up for 12 to 60 months with a mean of 28.5 months, without internal fixation loosening or breakage. There was significant differences in Cobb angle, vertebral anterior border height and recovery of spinal canal encroachment between preoperative and postoperative instantly (P < 0.05), however, there was no significant difference between postoperative instantly and final follow-up (P > 0.05). Thirteen cases obtained fusion by trans-vertebra fixation, 20 cases obtained fusion by intra-vertebra fixation, and 20 cases were treated by the combination of anterior and posterior approaches or one-stage posterior approaches all of patients obtained fusion. Spinal nervous function recovered I to II grade, 1 case was grade C, 3 cases were grade D, 52 cases were grade E. Localized pain was assessed as P1 in 52 cases, P2 in 3 cases, and P3 in 1 case. Working status was classified into W1 in 12 cases, W2 in 39 cases, and W3 in 5 cases. CONCLUSION: The lower lumbar vertebra and thoracolumbar junction exhibit different injury characteristics due to variations in anatomy and biomechanics. A comprehensive score of the AO classification, posterior column injury and degree of spinal canal encroachment will guide the selection of surgical method and approach for the treatment of lower lumbar vertebra burst fractures.

[Direct repair of adolescent lumbar spondylolysis using a pedicle screw-laminar hook system by paramedian approach].

OBJECTIVE: To discuss the indication and clinical effect of direct repair of adolescent lumbar spondylolysis by screw-laminar hook system. METHODS: From August 2003 to December 2008, 28 patients (13 males and 15 females,ranging in age from 15 and 26 years, averaged 21.6 years) with lumbar spondylolysis were treated with isthmic bone grafting and internal fixation with a pedicle screw-laminar hook system. Three patients had spondylolysis at L3, L4; 5 patients had spondylolysis at L4, L5; 8 patients had spondylolysis at L4; and 12 patients had spondylolysis at L5. All the patients had low back pain and lasted over 6 months. According to preoperative and postoperative plain radiograph, CT scan and Macnab criteria, the fusion rate and clinical effect of this technique were evaluated. RESULTS: All the patients were followed up with a mean period of 14.9 months, ranging from 9 to 24 months. All the patients had bony union according to the X-rays and CT scan. According to the calculation results of Macnab criteria, 22 patients got an excellent result, 5 good and 1 fair. CONCLUSION: The direct repair of adolescent lumbar spondylolysis with pedicle screw-laminar hook system can shorten length of operation,decrease blood loss, preserve more posterior structures of spine and avoid iatrogenic instability of spine. The postoperative immediate stability of vertebral segment is acquired and the mobility of adjacent intervertebral discs is reserved. The screw-laminar hook system for the treatment of adolescent spondylolisthesis can get satisfactory clinical results.

[Suffocation point and diurnal metabolism pattern of Hemifusus tuba (Gmelin)].

The individuals of Hemifusus tuba (Gmelin) were divided into 3 groups, i. e., small [S, (6.48 +/- 0.46) cm], medium[ M, (7.59 +/- 0.41) cm], and large [L, (9.08 +/- 0.37) cm], according to their shell height, and their suffocation points and diurnal metabolism patterns were investigated at water temperature (22 +/- 0.5) degrees C. The results indicated that the oxygen consumption rate of H. tuba was relatively stable and maintained at 1.81 mg x g(-1) x h(-1) when dissolved oxygen (DO) content was higher than 4.37 mg x L(-1), but decreased with decreasing DO when DO content was lower than 4.37 mg L(-1) x 0.43 mg L(-1) of DO was the suffocation point of H. tuba, with the oxygen consumption rate being 0. The standard metabolism (SM) and routine metabolism (RM) of H. tuba decreased significantly with increasing body mass, and changed with the same pattern in the 3 groups, i. e., being higher at night than in daytime. There was a significant difference in the SM (F = 36.263, P < 0.01) and RM (F = 6.788, P < 0.01) among the 3 groups. The peak values of the specific dynamic metabolism of groups S, M, and L were 2.11, 1.62, and 1.42 mg x g(-1) h(-1), being 1.09, 0.75, and 0.71 times higher than their SM, respectively, and maintained about 15 h. The ammonia excretion rates of groups S, M, and L reached the peak after 24, 24, and 27 hours of feeding, with the peak values being 3.94, 2.64, and 1.71 micromol x g(-1) x h(-1), and 0.87, 0.73 and 0.31 times higher than those in starvation state, respectively,.