In recent years,more and more of the enzyme proteins have been carried out using recombinant microorganism bioreactor for large scale production.For reasons of improved the catalytic capability and environmental suitability,or enhanced expression level of the protein,a variety of genetic engineering technology according to protein molecule modification have been applied extensively.Major strategies and achievements of molecular modification for microbial enzyme,such as site-directed mutagenesis,error-prone PCR,DNA shuffling and optimum codon design were reviewed.

Objective Liver dysfunction is a common complication of hyperthyroidism [ mainly Graves’ disease(GD)], that may restrict the choice as well as affect the ultimate outcome of treatment. The purpose of this study was to describe the clinical and biochemical patterns in patients suffering from Graves’ disease and liver dysfunction and to determine influential factors. Methods A total of 1 928 patients received radioactive iodine, 131 I treatment. Before 131 I therapy, 24 h radioactive iodine uptake of thyroid(24 h RAIU), serum free triiodothyronine (FT3 ), free thyroxine( FT4 ), sensitive thyroid-stimulating hormone( sTSH), anti-thyrotrophin receptor antibody (TRAb), thyroglobulin antibody(TgAb), anti-thyroid peroxidase antibody(TPOAb), and serum hepatic function parameters etc were performed. Data were analyzed by the unpaired t-test, the independent samples t-test, the χ2 test, logistic regression, and Pearson bivariate correlation. Results Ages, the course of Graves’ disease, the weight of thyroid, FT4 , TPOAb, and TRAb in Graves’ disease patients complicated with liver dysfunction were higher than those in patients with normal hepatic function, as shown in table 1. The influential factors including age, course of Graves’ disease, heart rate, weight of thyroid, FT4, 24 h RAIU, TgAb, TPOAb, and TRAb. 24 h RAIU were the protecting factors. Age, course of Graves’ disease, heart rate, weight of thyroid, FT4 , TRAb, and TPOAb were the risk factors. Conclusion The risk of liver dysfunction in patients with Graves’ disease was increased in the following cases: age over 45 years, heart rate above 90 bpm, weight of thyroid more than 35 g, course of Graves’ disease longer than 3 years, FT4 greater than 70. 5 pmol/ L, TPOAb above 360 IU/ ml, and TRAb above 15 IU/ L. In these coses 131 I therapy will be recommended.

Objective To report our experience of retroperitoneal laparoscopic extravascular stent placement for nutcracker syndrome.Methods The clinical data of 12 nutcracker syndrome patients (10 males and 2 females;mean age 26 years) who underwent retroperitoneal laparoscopic extravascular stent placement from March 2014 to Febuary 2016 were retrospectively reviewed.The main symptoms were gross hematuria in 8 patients(one with proteinuria)and flank pain was noted in 1 patient.Three male patients had left-sided secondary varicoceles.Ultrasonography and computed tomography showed the left renal vein clamped by the superior mesenteric artery and the aorta.The anteroposterior diameter of the left renal vein in the renal hilum was three-fold than the aortomesenteric area,and the peak velocity ratio of the aortomesenteric area was much faster than the renal hilum.Twelve patients underwent laparoscopic extravascular stent placement under general anesthesia.The preaortic fibrous tissue between the aorta and the superior mesenteric artery was released intraoperatively.Renal vein became fiat when the superior mesenteric artery was elevated.The 6-8 cm extravascular stent was set on the surface of the renal vein to prevent the compression.Results Stenting was successfully accomplished in all 12 patients.Mean operative time was 62 min (50-125 min),estimated blood loss was 35 ml(20-100 ml),and the hospital stay after operation was 8 days (6-12 days).Three patients had a transient orthostatic intolerance,and they were cured by conservative treatment.With a mean follow up of 14 months (5-30 months),symptoms of hematuria and flank pain resolved in 7/8 and 1/1,respectively.Varicoceles were cured in all three patients.One case got partial relief because of recurrent hematuria due to excessive exercise.Ultrasonography showed that extravascular stent was in the right place,and the angle between abdominal aorta and superior mesenteric artery became normal.The inner diameter of left renal vein was decreased,and the narrow segment was diminished in diameter meanwhile the blood outflow was smooth.Conclusions Retroperitoneal laparoscopic extravascular stent placement in the renal vein is a safe and effective approach for nutcracker syndrome.

Objective:To explore the impact of urinary iodine concentration (UIC) on response to 131I treatment in differentiated thyroid cancer (DTC) patients with different risk stratifications. Methods:A total of 181 patients with DTC (75 males, 106 females, age: (44.1±12.5) years), who received the first 131I treatment in Tianjin Medical University General Hospital between January 2018 and February 2019, were retrospectively analyzed. Patients were divided into low- to intermediate-risk and high-risk groups. The treatment response was categorized into excellent response (ER) and non-excellent response (non-ER). Factors being evaluated including age, sex, preablative stimulated thyroglobulin (ps-Tg), UIC, etc. Mann-Whitney U test, χ2 test and logistic regression analysis were used for data analysis. Results:The UIC and ps-Tg in the low- to intermediate-risk group ( n=113) was 111.60(55.80, 204.65) μg/L and 2.08(0.63, 4.91) μg/L, respectively. Compared with the ER subgroup ( n=86), non-ER subgroup ( n=27) had higher UIC and ps-Tg level ( z values: -2.585, -4.511, both P<0.05). In the high-risk group ( n=68), UIC was 115.40(61.23, 167.28) μg/L and ps-Tg was 16.65(4.52, 43.45) μg/L. Compared with the ER subgroup ( n=20), non-ER subgroup ( n=48) had higher ps-Tg level ( z=-4.677, P<0.01), while the UIC was not significantly different between ER and non-ER subgroups ( z=-0.013, P>0.05). The multivariate logistic analysis indicated the ps-Tg level was the significant variable for non-ER in low- to intermediate-risk group (odds ratio( OR)=6.157(95% CI: 1.046-36.227); OR=22.965(95% CI: 3.591-146.857), both P<0.05) and high-risk group ( OR=9.696 (95% CI: 1.379-68.169), P<0.05); a high UIC could be an indicator of non-ER only in the low- to intermediate-risk group ( OR=3.715(95% CI: 1.201-11.488), P<0.05). Conclusions:The non-ER is associated with UIC in the low- to intermediate-risk group; however, UIC does not affect the non-ER in the high-risk group. Higher ps-Tg level is associated with non-ER in patients with low- to intermediate-risk and high-risk DTC.

Objective To observe the mechanisms of autophagic eukaryotic cells in Acinetobacter microvilli removal and protein histological study on apoptosis induced by macrophages .Methods A model of Acinetobacter baumannii infection was established in 24 female OCR mice .The mice were randomly divided into control group (n= 12) and observation group (n= 12) .The control group was injected with normal saline ,and the observation group was injected with autophagy eukaryotic cells ,the histopathological changes of Acinetobacter and the induction of macrophage apoptosis were observed .Results There was no significant difference in the bacterial counts between the two groups of mice immediately after implantation (P>0 .05) ,the bacterial counts in the 24 and 48 h in the observation group was significantly lower than that in the control group (P<0 .05) .The lung tissue of mice in the ob-servation group injected after autophagy was normal ,the alveolar cavity was open ,no abnormal substances were found ,the alveolar wall was not obviously thickened ,and no inflammatory cell infiltration was found in the wall .The mice in the control group were in-jected with normal saline and lacked the ability to remove Acinetobacter ,resulting in a large number of inflammatory cell infiltra-tion ,vasodilatation ,and congestion in some mice .Conclusion Autophagic eukaryotic cells injected with Acinetobacter baumannii can increase the clearance rate ,induce apoptosis of macrophages and improve the quality of Acinetobacter baumannii .

Objective:To investigate the prognostic factors of differentiated thyroid cancer (DTC) patients with positive thyroglobulin antibody (TgAb) and varying ages after operation and 131I treatment. To explore the value of TgAb level and its change in the prognosis of DTC patients. Methods:Clinical data of 131 TgAb positive DTC patients were retrospectively analyzed. According to age, they were divided into young group(age<55 years, n=95) and elder group (age≥55 years, n=36). According to response, it was divided into excellent response group (110 cases) and non-excellent response group (21 cases). χ2 test and t test were used to compare the clinicopathological features between excellent response group and non-excellent response group. By logistic regression analysis, the independent risk factors affecting the prognosis of patients were analyzed. The receiver operating characteristic curve was used to determine the TgAb value of persistent or recurrent DTC, and the Kaplan-Meier regression curve was used to analyze the time of TgAb becoming negative. P<0.05 was statistically significant. Results:In young patients, the higher serum TgAb level before 131I treatment and the lateral lymph node metastasis were the independent influencing factors of poor prognosis [ OR=0.89(95% CI 0.83-0.95), OR=0.15(95% CI 0.05-0.52); both P<0.05]. In elder group, extraglandular invasion and higher serum TgAb before 131I treatment were associated with poorer prognosis [ OR=0.05(95% CI 0-0.83), OR=0.91(95% CI 0.76-1.13); P<0.05]. The serum TgAb thresholds for predicting DTC persistence/recurrence were 315.5 IU/mL(246.0 IU/mL in the young group and 516.5 IU/mL in the elder group). The mean time TgAb sera turned negative was (26.37±2.22) months [(23.28±2.37) months for young group and (32.64±4.07) months for elder group]. The TgAb decreased >50% in one year of the patients who had a lower probability of disease persistence/recurrence than the group without ( P<0.05). Conclusions:The high level of serum TgAb before 131I treatment and lateral lymph node metastasis were independent factors of poor prognosis in young patients, while in elder patients, extraglandular tumor invasion and the high level of serum TgAb before 131I treatment were independent factors of poor prognosis. The rate of TgAb change one year after treatment may be used as an early marker for predicting the disease status of TgAb positive patients.

Objective:To analyze the short-term effect of targeted drugs on quality of life in patients with radioactive iodine-refractory differentiated thyroid cancer (RAIR-DTC).Methods:From February 2020 to April 2022, 19 RAIR-DTC patients (10 males, 9 females; age (54.5±14.5) years) who received targeted drugs therapy (sorafenib, lenvatinib or anlotinib) in Tianjin Medical University General Hospital were prospectively enrolled. The thyroglobulin (Tg) levels prior and 1, 3, 6 months post the targeted treatment, and the adverse events were measured and recorded. Response evaluation criteria in solid tumors (RECIST) 1.1 version was used to evaluate the treatment response. The quality of life based on five-level EuroQol five-dimensional questionnaire (EQ-5D-5L) was monitored prior and 3 months post the targeted treatment, and the prevalence rates of mobility, self-care, usual activities, pain/discomfort, and anxiety/depression were analyzed, and the scores of health assessment were assessed. Paired t test, Kruskal-Wallis rank sum test and χ2 test were used to analyze data. Results:The prevalence rates of mobility (8/19), self-care (6/19), usual activities (10/19), pain/discomfort (10/19), and anxiety/depression (12/19) in 3 months post treatment were higher than those prior treatment (1/19, 1/19, 1/19, 2/19, 2/19; χ2 values: 4.38-11.31, all P<0.05). The score of health assessment prior treatment was (84.37±6.25), which was higher than that at 3 months post treatment (71.63±9.14; t=5.02, P=0.001). After targeted treatment, 10 patients were with skin toxicity, 8 patients were with hypertension, 8 patients were with weight loss, 7 patients were with diarrhea, 6 patients were with fatigue, 5 patients were with hepatic dysfunction, 2 patients were with proteinuria, 2 patients were with muscle pain and 1 patient was with oral ulcer. Of 19 patients, 17 insisted on continuing treatment, and the other two stopped treatment. The Tg levels at 1, 3 and 6 months post treatment were 56.26(44.60, 210.50), 53.36(41.25, 203.07) and 54.35(34.71, 223.52) mg/L, respectively, which were lower than the level prior treatment with no significant difference (110.16(49.63, 294.50) mg/L; H=2.42, P=0.490). After 3 months of targeted treatment, the progression-free survival (PFS) rate was 16/17, including 7 patients with partial response (PR), 9 patients with stable disease (SD), and 1 patient with progression of disease (PD). After 6 months of targeted treatment, the PFS rate was 10/17, including 5 patients with PR, 5 patients with SD, and 7 patients with PD. Conclusion:After 3-6 months of targeted treatment, the tumor markers of most patients are decreased with metastases improved, but the adverse events of targeted drugs have a great impact on quality of life in patients with RAIR-DTC.

Objective To construct a model of Graves'disease ( GD ) with ( or ) Graves'ophthalmopathy ( GO) in BALB/c mice by immunization with pcDNA3. 1/TSHR289. Methods pcDNA3. 1/TSHR289 was injected into the bilateral gastrocnemius muscle of 35 model mice and electroporation was immediately performed. 10 control mice were injected with sterile saline and electroporated, while 5 blank mice were injected with sterile saline only. Each group of mice was immunized at 1, 4, 7, and 10 weeks, respectively. Serum total T4 , TSH, TSAb, and TSBAb were measured before immunization, 2 weeks after each immunization, as well as 5 and 8 weeks after the last immunization. CT scan was used to evaluate the morphological changes of the eyes of the mice.99m TcO4- imaging was used to measure the thyroid uptake function, and the pathological changes of the thyroid and orbital tissues were evaluated by HE staining. Results After the 2nd time immunization, the serum concentrations of TT4 , TSAb and TSBAb in GD mice were significantly higher than those of control and blank groups( F=13.781, 31.435, 36.112, P<0.01, respectively).The TSH continued to be significantly lower than that of control and blank groups(F=13.966, P<0.01) . After the 4th time immunizations, the ability of uptaking99m TcO4- in GD mice thyroid was significantly enhanced compared with the control group. The thyroid goiter with a large amount of lymphocyte infiltration, and the thyroid follicle was thin. CT scan of GO mice showed thickening and swelling of the extraocular muscles, and no abnormalities in tendon and muscle attachment points. HE staining showed thickening of extraocular muscle fibers, lymphocyte infiltration of extraocular muscles and orbital tissue, increased hyaluronic acid, and infiltration of fat cells. Conclusion GD or GO model can be successfully induced by multiple intramuscular injection of pcDNA3.1/TSHR289 in BALB/c mice.

Anti-thyroid drug (ATD), radioactive iodine (RAI) and thyroidectomy are treatment options for Graves disease (GD). Treatment strategies for Graves ophthalmopathy (GO) patients include thyroid function control, oral or intravenous corticosteroids, orbital radiotherapy or orbital decompression surgery. However, current treatments for GD and GO are also less ideal because they target the signs and symptoms rather than the pathogenic mechanisms. The development of treatment strategies that targeting the thyroid-stimulating hormone receptor (TSHR) or insulin-like growth factor 1 receptor (IGF-1R) alone or in combination may yield effective and better tolerated treatments for GD and GO. This paper reviews the progress and limitations of the 2 methods.

Objective:To explore new methods of treating Graves′ disease (GD) by targeting thyroid stimulating hormone receptor (TSHR) and intercellular adhesion molecule-1 (ICAM-1).Methods:The small interfering RNA (siRNA) targeting TSHR and the ICAM-1 monoclonal antibody (mAb) were designed and synthesized. Thirty GD model mice were randomly divided into siRNA treatment group, ICAM-1 mAb treatment group, and untreated GD group (10 mice in each group), and 10 normal mice were taken as blank control. Serum thyroxine (T 4), thyroid stimulating hormone (TSH), TSH receptor-stimulating antibody (TSAb) and TSH-stimulation blocking antibody (TSBAb) were measured before and after treatment. At the end of the treatment, body mass and heart rate of mice in each group were measured, and thyroid uptake of 99Tc mO 4-, thyroid size and pathological changes were evaluated. Independent-sample t test, paired t test and one-way analysis of variance were used to analyze data. Results:After three treatments, the body mass of mice in siRNA group and ICAM-1 mAb group were significantly lower than that of normal mice ( F=3.50, P=0.025); the heart rates of the mice in two groups were significantly lower than that of untreated GD mice ( F=24.73, P<0.001). Heart rate of mice treated with siRNA decreased significantly, close to that of normal mice. After treatment, the serum T 4((27.58±1.94) vs (65.71±6.89) μg/L, (27.24±3.50) vs (70.84±8.46) μg/L), TSAb ((331.44±43.38) vs (457.33±45.85) mU/L, (275.16±45.80) vs (443.91±42.32) mU/L) and TSBAb ((13.94±1.11) vs (15.83±5.92) mU/L, (14.59±1.02) vs (17.05±6.16) mU/L) levels of mice in both siRNA group and ICAM-1 mAb group significantly decreased ( t values: 4.45-10.87, all P<0.05), while the serum TSH levels of mice in two groups significantly increased ((0.13±0.05) vs (0.04±0.05) mU/L, (1.46±0.34) vs (0.06±0.03) mU/L; t values: -2.22, -5.87, P values: 0.007, <0.001). The elevated TSH level and decreased TSAb level of mice treated with ICAM-1 mAb were significantly different from those treated with siRNA ( t values: 1.03, -1.63, P values: 0.002, 0.031). After treatment, the uptake of 99Tc mO 4- in part of the thyroid lobes of mice was decreased, and the enlargement degree of the corresponding lobes was reduced. The thyroid pathology of mice in the treated groups showed that the absorption vacuoles of thyroid follicles were reduced, and the phenomenon of thinner colloids was improved. No obvious damage was observed in the heart, liver and kidneys of the mice. Conclusions:Both the siRNA targeting TSHR and ICAM-1 mAb have therapeutic effects on GD model mice. The siRNA is better at controlling heart rate, and ICAM-1 mAb is better at increasing TSH and decreasing TSAb. Each of the above treatment methods is safe and effective, which can provide new ideas for GD targeted therapy.