Design, synthesis, and biological evaluation of pyrido[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine derivatives as novel EGFRL858R/T790M inhibitors.

EGFR mutations have been identified in 20,000 reported NSCLC (non-small cell lung cancer) samples, and exon 19 deletions and L858R mutations at position 21, known as "classical" mutations, account for 85-90% of the total EGFR (epidermal growth factor receptor) mutations. In this paper, two series of EGFR kinase inhibitors were designed and synthesised. Among them, compound B1 showed an IC50 value of 13 nM for kinase inhibitory activity against EGFRL858R/T790M and more than 76-fold selectivity for EGFRWT. Furthermore, in an in vitro anti-tumour activity test, compound B1 showed an effective anti-proliferation activity against H1975 cells with an IC50 value of 0.087 µΜ. We also verified the mechanism of action of compound B1 as a selective inhibitor of EGFRL858R/T790M by cell migration assay and apoptosis assay.

Design, synthesis and biological evaluation of aminopyrimidine derivatives bearing a 4,5,6,7-tetrahydrothieno [3,2-c]pyridine as potent EGFR inhibitors.

To resolve the problem of drug resistance caused by epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer, we used the principle of collocation to design and synthesize a series of aminopyrimidine derivatives with 4,5,6,7-tetrahydrothieno [3,2-c]pyridine side chains (according to the binding mode of AZD9291 to EGFRT790M) for use as EGFRL858R/T790M kinase inhibitors. The most promising compound A12, a non-covalently bound reversible inhibitor, showed excellent kinase inhibitory activity against EGFRL858R/T790M, with an IC50 value of 4.0 nM and more than 42-fold selectivity for EGFRWT (IC50 = 170.0 nM). Moreover, compound A12 showed strong anti-proliferative activity against H1975 cells, with IC50 value of 0.086 µΜ. Additionally, the effective inhibition of cell migration and the promotion of apoptosis by A12 verified its mechanism of action, as a selective inhibitor of EGFRL858R/T790M.

Integrated Skin Transcriptomics and Serum Multiplex Assays Reveal Novel Mechanisms of Wound Healing in Diabetic Foot Ulcers.

Nonhealing diabetic foot ulcers (DFUs) are characterized by low-grade chronic inflammation, both locally and systemically. We prospectively followed a group of patients who either healed or developed nonhealing chronic DFUs. Serum and forearm skin analysis, both at the protein expression and the transcriptomic level, indicated that increased expression of factors such as interferon-γ (IFN-γ), vascular endothelial growth factor, and soluble vascular cell adhesion molecule-1 were associated with DFU healing. Furthermore, foot skin single-cell RNA sequencing analysis showed multiple fibroblast cell clusters and increased inflammation in the dorsal skin of patients with diabetes mellitus (DM) and DFU specimens compared with control subjects. In addition, in myeloid cell DM and DFU upstream regulator analysis, we observed inhibition of interleukin-13 and IFN-γ and dysregulation of biological processes that included cell movement of monocytes, migration of dendritic cells, and chemotaxis of antigen-presenting cells pointing to an impaired migratory profile of immune cells in DM skin. The SLCO2A1 and CYP1A1 genes, which were upregulated at the forearm of nonhealers, were mainly expressed by the vascular endothelial cell cluster almost exclusively in DFU, indicating a potential important role in wound healing. These results from integrated protein and transcriptome analyses identified individual genes and pathways that can potentially be targeted for enhancing DFU healing.

The multiple protective roles and molecular mechanisms of melatonin and its precursor N-acetylserotonin in targeting brain injury and liver damage and in maintaining bone health.

Melatonin is a neurohormone associated with sleep and wakefulness and is mainly produced by the pineal gland. Numerous physiological functions of melatonin have been demonstrated including anti-inflammation, suppressing neoplastic growth, circadian and endocrine rhythm regulation, and its potent antioxidant activity as well as its role in regeneration of various tissues including the nervous system, liver, bone, kidney, bladder, skin, and muscle, among others. In this review, we summarize the recent advances related to the multiple protective roles of melatonin receptor agonists, melatonin and N-acetylserotonin (NAS), in brain injury, liver damage, and bone health. Brain injury, including traumatic brain injury, ischemic stroke, intracerebral hemorrhage, subarachnoid hemorrhage, and newborn perinatal hypoxia-ischemia encephalopathy, is a major cause of mortality and disability. Liver disease causes serious public health problems and various factors including alcohol, chemical pollutants, and drugs induce hepatic damage. Osteoporosis is the most common bone disease in humans. Due in part to an aging population, both the cost of care of fracture patients and the annual fracture rate have increased steadily. Despite the discrepancy in the pathophysiological processes of these disorders, time frames and severity, they may share several common molecular mechanisms. Oxidative stress is considered to be a critical factor in these pathogeneses. We update the current state of knowledge related to the molecular processes, mainly including anti-oxidative stress, anti-apoptosis, autophagy dysfunction, and anti-inflammation as well as other properties of melatonin and NAS. Particularly, the abilities of melatonin and NAS to directly scavenge oxygen-centered radicals and toxic reactive oxygen species, and indirectly act through antioxidant enzymes are disscussed. In this review, we summarize the similarities and differences in the protection provided by melatonin and/or NAS in brain, liver and bone damage. We analyze the involvement of melatonin receptor 1A (MT1), melatonin receptor 1B (MT2), and melatonin receptor 1C (MT3) in the protection of melatonin and/or NAS. Additionally, we evaluate their potential clinical applications. The multiple mechanisms of action and multiple organ-targeted properties of melatonin and NAS may contribute to development of promising therapies for clinical trials.

The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis.

Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from the elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli-germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli-germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation.

Factor analysis of the Caregiver Quality of Life Index-Cancer (CQOLC) scale for Chinese cancer caregivers: a preliminary reliability and validity study of the CQOLC-Chinese version.

The English version of the Caregiver Quality of Life Index-Cancer (CQOLC) was translated into simplified Chinese (CQOLC-C), following cultural translation, back-translation and pretest steps. Three hundred and sixty one cancer caregivers participated in this study. Cronbach's alpha was used to assess CQOLC-C reliability. Exploratory factor analyses (EFA) was used to generate two models of the measure's factor structure, and confirmatory factor analyses (CFA) were used to test each model, such that the best model to explain the latent structure of the CQOLC-C was identified. EFA using different factor extraction methods yielded two models including four and eight factors. According to the CFA results, model 2 was better fit for the original study data, based on the RMSEA criterion [0.058(90% CI = 0.051-0.065)], χ2 (531) = 853.92, p < 0.0001; CFI (0.96), NNFI (0.96), IFI (0.97), and NFI (0.92). We also examined the effect of removing three items on the CQOLC-C factor structure and discuss the resulting differences from other versions. These results indicate that the CQOLC-C's factor structure does not fully fit the original theorized model. This study provides preliminary support for further use of the CQOLC-C. However, the present work provides only partial support for the relevance and construct validity of the scale for Chinese caregivers.

Association of maximum weight with hyperuricemia risk: a retrospective study of 21,414 Chinese people.

BACKGROUND: Obesity has been demonstrated to be associated with increased serum uric acid (SUA); however, little is known regarding the relationship between maximum weight, or maximum weight fluctuation, and uric acid concentration. Through retrospective means, we determined the association of maximum weight with SUA risk. METHODS: Data of 21,414 participants (8,630 males and 12,784 females) from the 2007-8 China National Diabetes and Metabolic Disorders Study were analyzed for parameters including lifestyle habits, biochemical blood analysis and self-reported maximum weight. RESULTS: Elevated SUA subjects shared a cluster of demographic features. After adjustment for age, gender, education, smoking, drinking, physical activity, WHR, height, eGFR(evaluate glomerular filtration rate), and diuretic usage, multivariate logistic regression models demonstrated maximum weight was associated with increased risk of elevated SUA level (P<0.001). Duration of maximum weight was related with decreased risk of elevated SUA level (P<0.001). There was a significant correlation between time of weight loss and risk of increased SUA level reduction (P<0.001). Furthermore, our data indicated that the degree of weight loss from maximum weight was another important factor for the risk of increased SUA level reduction (P<0.001). Finally, ROC curve analysis revealed area under the curve was 0.661 (95% CI, 0.647-0.674), statistically significant for maximum weight association with hyperuricemia (P<0.001). CONCLUSIONS: Maximum weight is a strong risk factor for increased uric acid level in the Chinese population, which might serve as a novel clinical indicator suggesting hyperuricemia. Controlling maximum weight, keeping weight to the appropriate range, and maintaining the stable weight may be conducive for decreasing risk of hyperuricemia.

Expression of EphrinB2 and EphB4 in glioma tissues correlated to the progression of glioma and the prognosis of glioblastoma patients.

OBJECTIVE: The ligand EphrinB2 and the corresponding receptor EphB4 are up-regulated and involved in tumour growth in various human cancers. However, little is known about how this receptor-ligand complex contributes to the progression of glioma. This prompted us to study the association between the expressions of EphrinB2 and EphB4, clinicopathological variables, and glioma patient outcome. METHODS: Immunohistochemical staining was performed to detect the expression patterns of EphrinB2 and EphB4 in the biopsies from 96 patients with primary gliomas. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of patients. RESULTS: Immunohistochemical analysis revealed that the expression of EphrinB2 was significantly correlated with that of EphB4 (r=0.86, p=0.002). EphrinB2 and EphB4 were significantly associated with the Karnofsky performance scale (KPS) score and World Health Organization grades of patients with gliomas, respectively. Especially, the positive expression rates of EphrinB2 and EphB4 were significantly higher in patients with higher grade (both p=0.001) and lower KPS score (p=0.002 and 0.003, respectively). Multivariate Cox regression analysis revealed that EphrinB2 and EphB4 expressions were both independent prognostic factors for progress-free survival of glioblastoma patients (both p=0.02). CONCLUSION: Our data indicated for the first time that EphrinB2 and EphB4 expressions increase according to the histopathological grade and KPS score of glioma, and their expression levels are related to the progression-free survival of glioblastoma patients.

Anti-diabetic activities of Acanthopanax senticosus polysaccharide (ASP) in combination with metformin.

Combination therapy had become very popular currently for the diabetes mellitus and its complications, because of long term unreasonable drug use and adverse reaction to human body. In this study, a polysaccharide (ASP) from the roots of Acanthopanax senticosus was evaluated as an adjuvant with metformin for antidiabetic therapy in alloxan-induced diabetic rats. The result identified ASP plus metformin had a more beneficial promotion for relieving the symptoms of diabetes and reversing liver and kidney damage to normal level than only metfomin administration to diabetic rats. The blood glucose, blood lipid (TC and TG), thiobarbituric acid reactive substances (TBARS), AST, ALT, ALP, total bilirubin, creatinine and urea levels in diabetic rats were decreased by combination of ASP and metformin. Furthermore, the body weight, liver glycogen formation, antioxidant substance (GSH) and antioxidant enzyme (SOD and GPX) levels increased evidently in diabetic mice treated with both ASP and metformin. In particular, sometimes ASP plus metformin could significantly reverse the pathophysiologic parameters of diabetic rats to normal level than only metformin administration. Therefore ASP could be developed to a new adjuvant combined with metformin for diabetes mellitus therapy in the future.

Unbalanced MMP/TIMP-1 expression during the development of experimental pulmonary fibrosis with acute paraquat poisoning.

Paraquat (PQ)-induced pulmonary toxicity is known to result in pulmonary edema, infiltration of inflammatory cells and damage to the alveolar epithelium, which may progress to severe fibrosis. Matrix metalloproteinases (MMPs) and their physiological inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs), which degrade and remodel the excess extracellular matrix, are believed to play an important role in the development of fibrotic tissue. In this study, we examined the sequential expression of MMP-2, MMP-9 and TIMP-1 in a rat model of pulmonary fibrosis induced by PQ. Adult male Sprague-Dawley rats were treated intraperitoneally with PQ (20 mg/kg) and saline (control group). Rats were sacrificed at days 1, 3, 7 and 21 after the PQ treatment. Lungs were excised for histological evaluation and immunohistochemical analyses, as well as the determination of collagen content, gene expression by fluorimeter-based quantitive RT-PCR assay and gelatinolytic activity by zymography. Lung MMP-2 and -9 mRNA expression progressively increased and reached a peak on day 7 after PQ treatment, while TIMP-1 mRNA levels in the PQ-treated lungs reached a peak on day 21 after modeling. Lung zymography revealed an increase in progelatinase B, progelatinase A and their active forms. In conclusion, unbalanced MMP/TIMP-1 expression and excessive gelatinolytic activity contribute to PQ-induced pulmonary fibrosis. Their precise role should be studied in depth as they may represent relevant therapeutic targets for PQ poisoning-induced pulmonary fibrosis.

Activation of JAK/STAT signal pathway predicts poor prognosis of patients with gliomas.

JAK/STAT pathway transmits signals from the cell membrane to the nucleus in response to extracellular growth factors and cytokines. Activation of this pathway has been found in certain types of human tumors. The goal of this study was to investigate the correlation between the JAK/STAT pathway in human gliomas and patients' prognosis, which currently is unknown. Western blotting analysis and immunohistochemical staining were performed to detect JAK-1, phosphorylated JAK-1, and STAT-3 expression patterns in the biopsies from 96 patients with primary gliomas. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of patients. Western blotting analysis and immunohistochemical staining both indicated that the expression levels of JAK-1, phosphorylated JAK-1, and STAT-3 in primary glioma tissues were significantly higher than those in normal brain tissues (P < 0.001). Especially, the positive expression rates of JAK-1, phosphorylated JAK-1, and STAT-3 were significantly higher in patients with higher grade (P = 0.001, 0.001, and 0.002, respectively) and lower KPS score (P = 0.01, 0.008, and 0.01, respectively). Statistical analysis showed that patients with gliomas expressing JAK-1 and STAT-3 have lower overall survival rates relative to those not expressing these proteins. Cox multi-factor analysis showed that KPS (P = 0.03), WHO grade (P = 0.008), JAK-1 (P = 0.005), and STAT-3 (P = 0.006) were independent prognosis factors for human gliomas. These results provide convincing evidence for the first time that the JAK/STAT pathway may play a role in the progression of human gliomas. Its activated state might be a potent tool for predicting the clinical prognosis of patients with glioma.

CD147 and VEGF co-expression predicts prognosis in patients with acute myeloid leukemia.

OBJECTIVE: To investigate the possible role of CD147 and vascular endothelial growth factor in progression and prognosis of acute myeloid leukemia. METHODS: Immunohistochemical staining was performed to detect the expression of CD147 and vascular endothelial growth factor in paraffin-embedded sections from 62 bone marrow biopsies obtained from an equal number of patients with newly diagnosed acute myeloid leukemia. RESULTS: CD147 and vascular endothelial growth factor expression in the bone marrow of acute myeloid leukemia patients were significantly higher than those in normal controls (both P < 0.001). Expression of them was significantly increased in patients with a high degree of microvessel density compared with those with a low degree (CD147: P = 0.009; vascular endothelial growth factor: P = 0.01) and correlated well with bone marrow microvessel density (CD147: P = 0.01; vascular endothelial growth factor: P = 0.02). In addition, higher levels of CD147 and vascular endothelial growth factor were also found in acute myeloid leukemia patients with an unfavorable karyotype compared with those with intermediate and favorable karyotypes (both P = 0.01). Moreover, the expression of CD147 was significantly correlated with that of vascular endothelial growth factor (P < 0.001). Furthermore, the co-expression of CD147 and vascular endothelial growth factor in the bone marrow indicated a poor prognosis in acute myeloid leukemia and was an independent prognostic factor for overall survival by multivariate analysis. CONCLUSIONS: Our data show for the first time that the co-expression of CD147 and vascular endothelial growth factor may indicate a poor prognosis in acute myeloid leukemia and may be a highly sensitive marker for predicting the clinical outcome of patients.

Expression patterns of ClC-3 mRNA and protein in aortic smooth muscle, kidney and brain in diabetic rats.

ClC-3, a member of the ClC family of voltage-gated chloride channels, regulates cell proliferation of cultured rat aortic vascular smooth muscle cells, pathogenesis of allergic rhinitis and tumor cell migration. However, its role in diabetic animals is still unknown. To address this issue, we investigated the expression patterns of ClC-3 in diabetic rats. Five-week-old Sprague-Dawley rats were divided into two groups, 50 non-diabetic control rats (non-DM) and 50 diabetic model rats (DM). ClC-3 mRNA and protein expression in aortic smooth muscle, kidney and brain tissues were examined by fluorimeter-based quantitive RT-PCR assay and Western blot analysis, respectively. ClC-3 mRNA and protein were endogenously expressed in aortic smooth muscle, kidney (cortex and medulla) and brain tissues of both control and streptozotocin-induced diabetic rats. ClC-3 mRNA and protein expression levels were significantly higher in aortic smooth muscle and brain tissues of diabetic rats, but significantly decreased in kidney medulla tissue, relative to non-DM controls. There were no significant differences in ClC-3 mRNA and protein expression in kidney cortex between non-diabetic control and diabetic rats. Furthermore, the altered ClC-3 expression patterns in diabetic rat aortic smooth muscle, brain, and kidney medulla tissues all correlated with the changes in blood glucose levels (p < 0.05). In conclusion, our data show for the first time that diabetes alters both the gene and protein expression of ClC-3 channels. These changes may contribute to the impaired vascular, brain and kidney functions observed in diabetes.

Over-expression of neuroepithelial-transforming protein 1 confers poor prognosis of patients with gliomas.

OBJECTIVE: Neuroepithelial-transforming protein 1 is a member of the guanine nucleotide exchange factor family, a group of proteins which are known to activate and thereby regulate Rho family members. Deregulation of neuroepithelial-transforming protein 1 expression has been found in certain types of human tumors. To investigate its prognostic value in human gliomas, which is currently unknown, we examined the correlation between neuroepithelial-transforming protein 1 expression and prognosis in patients with gliomas. METHODS: Immunohistochemical staining was performed to detect neuroepithelial-transforming protein 1 expression patterns in the biopsies from 96 patients with primary gliomas. Kaplan-Meier survival and Cox's regression analyses were performed to evaluate the prognosis of patients. RESULTS: Immunohistochemical analysis with anti-neuroepithelial-transforming protein 1 antibody revealed that neuroepithelial-transforming protein 1 was significantly associated with the Karnofsky performance scale score and World Health Organization grades of patients with gliomas. Especially, the positive expression rates of neuroepithelial-transforming protein 1 were significantly higher in patients with higher grade (P = 0.001) and lower Karnofsky's performance scale score (P = 0.005). The median survival of patients with high neuroepithelial-transforming protein 1 expression was significantly shorter than that with low expression and without expression (316, 892 and 1180 days, respectively). Cox's multifactor analysis showed that the Karnofsky performance scale (P = 0.01), World Health Organization grade (P = 0.008) and neuroepithelial-transforming protein 1 (P = 0.006) were independent prognosis factors for human glioma. CONCLUSIONS: Taken together, our study indicates for the first time that neuroepithelial-transforming protein 1 status may be a highly sensitive marker for glioma prognosis and suggest that the expression patterns of neuroepithelial-transforming protein 1 might be a potent tool for predicting the clinical prognosis of glioma patients.

Expression pattern and clinical prognostic relevance of bone morphogenetic protein-2 in human gliomas.

OBJECTIVE: Bone morphogenetic protein-2 (BMP-2) is normally expressed in the embryo promoting the development of several organs. Aberrant expression of BMP-2 occurs in various tumors. However, a correlation between BMP-2 expression in human gliomas and patients' prognosis has not been reported. To address this question, this study was to investigate the BMP-2 expression pattern in human gliomas and to evaluate its prognostic relevance. METHODS: We analyzed the expression of the BMP-2 antigen in a series of 98 gliomas of various grade and histology by immunohistochemistry on paraffin-embedded sections. Then, the correlation of BMP-2 expression pattern with clinical-pathological features of patients and its prognostic relevance were determined. RESULTS: Immunohistochemical analysis with anti-BMP-2 antibody revealed dense and spotty staining in the tumor cells and its expression levels became significantly higher as the gliomas' grade advanced (P < 0.001). The median survival of patients with intensively positive BMP-2 expression was significantly shorter than that with negative expression (318 vs. 1197 days, P < 0.0001). The Kaplan-Meier survival curves showed that the BMP-2 expression was not only a significant predictor of survival in high-grade gliomas (grade IV, P = 0.02), but also in lower-grade gliomas (grades II and III, P < 0.001). CONCLUSIONS: These results indicate that BMP-2 is a highly sensitive marker for gliomas prognosis and suggest that the expression level of BMP-2 may be a potent tool for the clinical prognosis of gliomas patients.

The correlation between intestinal gonadotropin-releasing hormone (GnRH) and proglucagon in hyperlipidemic rats and Goto-Kakizaki (GK) rats.

Our experiment investigated the mRNA expression of intestinal gonadotropin-releasing hormone (GnRH), proglucagon (PG), and glucagon-like peptide 1 receptor (GLP-1R) in the jejunum, ileum, and colon of rats fed with high-fat diet and Goto-Kakizaki (GK) rats and revealed the physiological role of intestinal GnRH. We found that the GnRH and PG mRNA levels in high-cholesterol (HCh) diet were higher than in the control. However, the GnRH receptor (GnRHR) and GLP-1R mRNA levels did not differ significantly between HCh and control. The GnRH, PG, and GLP-1R mRNA levels in GK rats were lower, respectively, than those in control rats, while the GnRHR levels did not differ significantly between GK rats and control rats. There were no difference in GnRH, PG, GnRHR, and GLP-1R mRNA levels in the ileum and colon tissue between HCh and control rats. The GnRH mRNA levels of GK rats were lower than those in control rats; however, the PG, GLP-1R, and GnRHR levels did not differ significantly between GK and control rats. The GLP-1R mRNA levels of GK rats were lower than those in control rats. The GnRH mRNA expression showed positive correlation with PG mRNA expression in different intestinal sections. The GnRH level in the jejunum showed a significant effect on blood glucose level, while the PG level in the jejunum showed a significant effect on insulin level. This may imply that, compared with the ileum and colon, the jejunum had greater impact on glucose metabolism; furthermore, GnRH might interact with intestinal GLP-1 and GLP-2 through the paracrine and autocrine ways and then regulate glucose metabolism and insulin secretion.

[Cloning and sequence analysis of variable region gene of anti-idiotype monoclonal antibody against Vibrio alginolyticus].

AIM: To clone and sequence V(H) and V(L) genes of anti-idiotype monoclonal antibody (mAb) against vibrio alginolyticus. METHODS: Total RNA was extracted from hybridoma cell AL1 secreting mAb against vibrio alginolyticus and cDNA was amplified by RT-PCR. Then the cDNA was inserted into PMD18-T vector and its sequence was analyzed. RESULTS: The V(H) gene contained 369 bp and encoded 123 amino acid residues; the V(L) gene contained 339 bp and encoded 113 amino acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) and V(L) genes, respectively. CONCLUSION: The successful cloning of the V(H) and V(L) genes of anti-idiotype mAb against vibrio alginolyticus provides a sound basis for construction of gene-engineering vaccine of the anti-idiotype mAb against vibrio alginolyticus.