Do antibody CDR loops change conformation upon binding?

Antibodies have increasingly been developed as drugs with over 100 now licensed in the US or EU. During development, it is often necessary to increase or reduce the affinity of an antibody and rational attempts to do so rely on having a structure of the antibody-antigen complex often obtained by modeling. The antigen-binding site consists primarily of six loops known as complementarity-determining regions (CDRs), and an open question has been whether these loops change their conformation when they bind to an antigen. Existing surveys of antibody-antigen complex structures have only examined CDR conformational change in case studies or small-scale surveys. With an increasing number of antibodies where both free and complexed structures have been deposited in the Protein Data Bank, a large-scale survey of CDR conformational change during binding is now possible. To this end, we built a dataset, AbAgDb, that currently includes 177 antibodies with high-quality CDRs, each of which has at least one bound and one unbound structure. We analyzed the conformational change of the Cα backbone of each CDR upon binding and found that, in most cases, the CDRs (other than CDR-H3) show minimal movement, while 70.6% and 87% of CDR-H3s showed global Cα RMSD ≤ 1.0Å and ≤ 2.0Å, respectively. We also compared bound CDR conformations with the conformational space of unbound CDRs and found most of the bound conformations are included in the unbound conformational space. In future, our results will contribute to developing insights into antibodies and new methods for modeling and docking.

Network analysis reveals dysregulated functional patterns in type II diabetic skin.

Skin disorders are one of the most common complications of type II diabetes (T2DM). Long-term effects of high blood glucose leave individuals with T2DM more susceptible to cutaneous diseases, but its underlying molecular mechanisms are unclear. Network-based methods consider the complex interactions between genes which can complement the analysis of single genes in previous research. Here, we use network analysis and topological properties to systematically investigate dysregulated gene co-expression patterns in type II diabetic skin with skin samples from the Genotype-Tissue Expression database. Our final network consisted of 8812 genes from 73 subjects with T2DM and 147 non-T2DM subjects matched for age, sex, and race. Two gene modules significantly related to T2DM were functionally enriched in the pathway lipid metabolism, activated by PPARA and SREBF (SREBP). Transcription factors KLF10, KLF4, SP1, and microRNA-21 were predicted to be important regulators of gene expression in these modules. Intramodular analysis and betweenness centrality identified NCOA6 as the hub gene while KHSRP and SIN3B are key coordinators that influence molecular activities differently between T2DM and non-T2DM populations. We built a TF-miRNA-mRNA regulatory network to reveal the novel mechanism (miR-21-PPARA-NCOA6) of dysregulated keratinocyte proliferation, differentiation, and migration in diabetic skin, which may provide new insights into the susceptibility of skin disorders in T2DM patients. Hub genes and key coordinators may serve as therapeutic targets to improve diabetic skincare.

The dynamic wound microbiome.

BACKGROUND: Diabetic foot ulcers (DFUs) account for the majority of all limb amputations and hospitalizations due to diabetes complications. With 30 million cases of diabetes in the USA and 500,000 new diagnoses each year, DFUs are a growing health problem. Diabetes patients with limb amputations have high postoperative mortality, a high rate of secondary amputation, prolonged inpatient hospital stays, and a high incidence of re-hospitalization. DFU-associated amputations constitute a significant burden on healthcare resources that cost more than 10 billion dollars per year. Currently, there is no way to identify wounds that will heal versus those that will become severely infected and require amputation. MAIN BODY: Accurate identification of causative pathogens in diabetic foot ulcers is a critical component of effective treatment. Compared to traditional culture-based methods, advanced sequencing technologies provide more comprehensive and unbiased profiling on wound microbiome with a higher taxonomic resolution, as well as functional annotation such as virulence and antibiotic resistance. In this review, we summarize the latest developments in defining the microbiology of diabetic foot ulcers that have been unveiled by sequencing technologies and discuss both the future promises and current limitations of these approaches. In particular, we highlight the temporal patterns and system dynamics in the diabetic foot microbiome monitored and measured during wound progression and medical intervention, and explore the feasibility of molecular diagnostics in clinics. CONCLUSION: Molecular tests conducted during weekly office visits to clean and examine DFUs would allow clinicians to offer personalized treatment and antibiotic therapy. Personalized wound management could reduce healthcare costs, improve quality of life for patients, and recoup lost productivity that is important not only to the patient, but also to healthcare payers and providers. These efforts could also improve antibiotic stewardship and control the rise of "superbugs" vital to global health.

Omega-3PUFA Attenuates MNU-Induced Colorectal Cancer in Rats by Blocking PI3K/AKT/Bcl-2 Signaling.

BACKGROUND: Omega 3 polyunsaturated fatty acid (Omega-3PUFA) is one of the essential nutrients for human body involved in intracellular metabolic regulation and cell signaling. Previous studies have shown that Omega-3PUFA is involved in the pathogenesis of digestive system tumors, including colorectal cancer (CRC), however, the effects of Omega-3PUFA on CRC has not been fully elucidated. In the current study, we evaluated whether Omega-3PUFA can alleviate N-methyl-N-nitrosourea(MNU) induced CRC in a rat model and illustrated the potential mechanism. METHODS: The effects of Omga-3PUFA on MNU-induced colorectal cancer in rats were analyzed by in vivo experiments. The viability, apoptosis, colony formation and invasion of CRC cells treated with Omga-3PUFA were detected by CCK8, flow cytometry, clone formation assay and transwell invasion assay. The expression of apoptosis-related proteins in CRC cells treated with Omga-3PUFA was detected by Western blotting. Finally, after adding PI3K activator, the viability, apoptosis and protein expression of CRC cells treated with Omga-3PUFA were detected by CCK8, flow cytometry and Western blotting. RESULTS: Our results showed that Omega-3PUFA attenuated MNU-induced CRC in rats and inhibited AKT/Bcl-2 signaling in rats. In addition, Omega-3PUFA inhibited CRC cell proliferation and induces CRC cell apoptosis. Moreover, Omega-3PUFA inhibited CRC cell colony formation and invasion, and inhibited PI3K/AKT/Bcl-2 signaling in CRC cells. Furthermore, The effects of Omega-3PUFA on cell proliferation and apoptosis were inhibited by blocking PI3K/AKT signaling. CONCLUSION: Omega-3PUFA can attenuate MNU-induced colorectal cancer in rats by blocking PI3K/AKT/Bcl-2 signaling, which suggests that Omega-3PUFA may be a potent agent for CRC treatment.

Omega-3 Polyunsaturated Fatty Acids Inhibited Tumor Growth via Preventing the Decrease of Genomic DNA Methylation in Colorectal Cancer Rats.

Omge-3 polyunsaturated fatty acids (PUFAs) exhibited significant effect in inhibiting various tumors. However, the mechanisms of its anticancer role have not been fully demonstrated. The declination of 5-methylcytosine (5 mC) was closely associated with poor prognosis of tumors. To explore whether omega-3 PUFAs influences on DNA methylation level in tumors, colorectal cancer (CRC) rat model were constructed using N-methyl phosphite nitrourea and omega-3 PUFAs were fed to part of the rats during tumor induction. The PUFAs contents in the rats of 3 experimental groups were measured using gas chromatography and 5 mC level were detected by liquid chromatography tandem mass spectrometry. The results showed that tumor incidence in omega-3 treated rats was much lower than in CRC model rats, which confirmed significant antitumor role of omega-3 PUFAs. Six PUFA members categorized to omega-3 and omega-6 families were quantified and the ratio of omega-6/omega-3 PUFAs was remarkably lower in omega-3 PUFAs treatment group than in CRC model group. 5 mC content in omega-3 PUFAs treated rats was higher than in CRC model rats, suggesting omega-3 PUFAs promoted 5 mC synthesis. Therefore, omega-3 PUFAs probably inhibited tumor growth via regulating DNA methylation process, which provided a novel anticancer mechanism of omega-3 PUFAs from epigenetic view.

[Determination of global DNA methylation in tissues by hydrophilic interaction liquid chromatography].

A hydrophilic interaction liquid chromatographic (HILIC) method has been developed for the determination of global DNA methylation in tissues. The DNA was extracted by phenol-chloroform, hydrolyzed with 88% formic acid at 140 degrees C, evaporated under nitrogen at 60 degrees C, and reconstituted in a mixture of acetonitrile/water (90:10, v/v); the separation was achieved on a Waters bridged ethylene hybrid (BEH) HILIC column (100 mm x 2.1 mm, 1.7 microm). The cytosine (Cyt) and 5-methylcytosine (5-mCyt) were separated in a fairly short time (3.5 min) by isocratic elution with a mixture of acetonitrile/10 mmol/L ammonium formate (94:6, v/v) as the mobile phase. Under the optimized conditions, calibration standard curve showed a good linearity in the range 1-900 micromol/L for Cyt and in the range 1-64 micromol/L for 5-mCyt with correlation coefficients of 0.9999 and 0.9998, respectively. The limit of detection (LOD) was 54 nmol/L (0.54 pmol on-column) both for Cyt and 5-mCyt, and the limit of quantification was 250 nmol/L (2.5 pmol on-column) both for Cyt and 5-mCyt. The recoveries of Cyt at the spiked levels of 90, 450, 900 micromol/L and 5-mCyt at the spiked levels of 5, 16, 64 micromol/L all ranged from 94.7% to 100.5% with a relative standard deviations less than 1.48%. The method was applied to the analysis of DNA from colon cancer tissue, and the average degree of methylation was 4.0%. The method is rapid, simple, sensitive, reliable, and suitable for the determination of global DNA methylation.

Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry.

We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol-chloroform, hydrolyzed using 88% formic acid at 140°C, spiked with cytosine-2,4-(13)C(15)N(2) as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC-MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1-50 and 1-100ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70-4.09% and 0.60-4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC-MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation.

[Protective effect of glutamine on intestinal barrier function in patients receiving chemotherapy].

OBJECTIVE: To study the protective effect of glutamine (Gln) on intestinal permeability in patients receiving chemotherapy. METHODS: Thirty-nine patients with gastrointestinal cancer after operation were randomly divided into Gln and control groups, and received oral administration of glutamine (30 g/d) for 7 days (n=22) or not (n=17). All patients received CF+ 5-FU chemotherapy for 5 days. Serum concentration of glutamine and urinary lactulose/mannitol (L/M) ratio were measured before and 1 day after chemotherapy. RESULTS: After chemotherapy, the serum Gln concentration was significantly decreased to (535.42+/- 53.75) micromol/L in the control group and increased to (54.44+/- 81.26) micromol/L in the Gln group, and there was significant difference between the two groups (P< 0.01). Urine L/M ratio was significantly increased to (0.0453+/- 0.0078) in the control group and decreased to (0.0331+/- 0.0061) in the Gln group, and there was significant difference between the two groups after chemotherapy (P< 0.01). CONCLUSION: Oral administration of glutamine granules can increase serum concentration of glutamine in chemotherapy patients with gastrointestinal cancer and can decrease intestinal permeability, maintain intestinal barrier.

[The clinical features of acute pancreatitis in 10 cities in Shandong Province].

OBJECTIVE: To investigate and obtain a more comprehensive view of the etiology and clinical features of acute pancreatitis (AP) in Shandong Province. METHODS: 1471 cases admitted to hospital for AP were studied and collected from the ten cities of Shandong Province from January 1992 to December 2002 retrospectively. Data of each enrolled patient was recorded in a standardized form. RESULTS: In the 1471 patients, the ratio of male: female was 854:617, and also the mean age of them and the range was 43.3 and from 13 - 82 years old. 1280 had mild AP, and 191 had sever AP. Cholelithiasis (20.2%), alcohol (17.3%) and diet-induced (12.4%) were the most frequent etiologic factors, followed by biliary tract infections (5.6%), hyperlipemia (2.3%), other factors (5.1%). But in about 36.1% cases, the etiology of AP still remains unexplained. In coastal regions, cholelithiasis is the most frequent factor but in interior regions alcohol ranked first. In male, a small predominance of alcohol over cholelithiasis was seen (27.4 vs.14.3%, P < 0.01); and in female, there was a clear predominance of cholelithiasis over alcohol (28.4 vs. 3.2%, P < 0.01). The complications of AP were pancreatic pseudocyst, ascites and peritonitis, pulmonary infections, multiple organ failure, diabetes mellitus-2 and shock, etc. according to their frequencies. CONCLUSIONS: Cholelithiasis, alcohol and diet-induced factor were main etiologic factors in Shandong Province, whereas cholelithiasis alone predominated in the females. In about 36.1% cases, the etiology remains unknown. So that more attention should be paid to study the etiology of AP.