Optimising infliximab induction dosing to achieve clinical remission in Chinese patients with Crohn's disease.

Aims: A strategy based on therapeutic drug monitoring and population pharmacokinetic (popPK) models would likely increase the rate of clinical remission (CR) after infliximab (IFX) induction in patients with Crohn's disease (CD). This study aimed to evaluate the relationship between early IFX levels and antibodies to infliximab (ATI) and CR at week 14 and simulate the probability of attaining the identified exposure target. Methods: Patients with CD (n = 140) treated with IFX were enrolled to develop the popPK model. Of these, 43 moderate-to-severe patients with CD were followed up at week 14. Simulations were performed on patients with different dosage regimens and covariates. Results: IFX levels >20.08 µg/mL at week 2, >18.44 µg/mL at week 6, and >3.08 µg/mL at week 14 were linked to CR. A one-compartment model fit the data best. The covariates influencing clearance were fat free mass, albumin and ATI levels. To achieve IFX levels >20.08 µg/mL at week 2, ≥400 mg IFX was predicted to be required in over 50% patients with 45-70 kg and 35-45 g/L albumin, except for patients with 70 kg and 30 g/L albumin. Conclusion: IFX levels >20.08 µg/mL at week 2 and absence of ATI at week 14 are associated with CR. Optimising IFX induction dosing will be critical to achieve the target of early IFX levels associated with CR.

Physiological and communicative emotional disconcordance in children on the autism spectrum.

BACKGROUND: Individuals on the autism spectrum commonly have differences from non-autistic people in expressing their emotions using communicative behaviors, such as facial expressions. However, it is not yet clear if this reduced expressivity stems from reduced physiological reactivity in emotional contexts or if individuals react internally, but do not show these reactions externally to others. We hypothesized that autism is characterized by a discordance between in-the-moment internal psychophysiological arousal and external communicative expressions of emotion. METHODS: Forty-one children on the autism spectrum and 39 non-autistic, typically developing (TD) children of two age groups (2-4 and 8-12 years) participated in a low-level stress task whilst wearing a wireless electrocardiogram. Children's negative emotional expressions (facial, vocal, bodily) were coded following standardized protocols. Alexithymia traits were assessed using the Children's Alexithymia Measure with school-aged children only. Data analyses involved ANOVAs, correlations, and sensitivity analyses. RESULTS: There were no group differences in physiological arousal (heart rate) or in communicative expressions of stress to the stress task. For TD preschoolers, physiological arousal during the stress task was associated with vocal expressions and for TD school-aged children, they were associated with facial and bodily expressions. By contrast, for children on the autism spectrum, physiological arousal during the stress tasks was not associated with communicative expressions across age groups. CONCLUSIONS: Our findings suggest that children on the autism spectrum might experience emotional disconcordance, in that their physiological arousal does not align with their communicative expressions. Therefore, the internally experienced stress of children on the autism spectrum may be inadvertently missed by teachers and caregivers and, consequently, learning opportunities for teaching emotional communication and regulation may be also missed. Our results support the use of wearable biosensors to facilitate such interventions in children on the autism spectrum.

Association between genetic variants and development of antibodies to infliximab: A cross-sectional study in Chinese patients with Crohn's disease.

Aims: Genetic variants increase the susceptibility to anti-drug antibodies (ADA) in response to anti-TNF therapy in chronic inflammatory diseases. However, little is known about genetic variants in Chinese populations. This study aimed to identify genetic variants contributing to the risk of the development of antibodies to infliximab (ATI) in Chinese patients with Crohn's disease (CD). Methods: CD patients (n = 104) treated with infliximab (IFX) during the maintenance therapy were enrolled in this cross-sectional study. ATI was assessed by an in-house developed drug-tolerant ELISA method. ATI titers of 1:20 and ≥1:60 were considered a low titer and a high titer, respectively. Thirteen types of single nucleotide polymorphisms (SNPs) within 13 genes involved in the immune process, the susceptibility to chronic inflammatory diseases, cytokines and apoptosis pathways were investigated. Results: The median trough levels of infliximab (TLI) in patients with clinical remission (CR) were higher than those in patients without CR (3.80 vs. 1.50 µg/mL, p < .001). The median TLI in patients with high-titer ATI was significantly lower than that in ATI-negative patients (1.15 vs. 4.48 µg/mL, p < .001) or those with low-titer ATI (1.15 vs. 2.95 µg/mL, p = .03). The HLA-DQA1*05 rs2097432 GG and GA genotypes were more frequent in patients with ATI (GG and AG vs. AA, 27/38 = 71.05% vs. 29/66 = 43.94%, OR 2.94, 95% CI 1.19-7.30, p = .02). Patients carrying the CC and AC genotypes of rs396991 in FCGR3A were associated with a higher frequency of ATI formation (CC and AC vs. AA, 37/57 = 64.91% vs. 19/47 = 40.43%, OR 2.94, 95% CI 1.24-6.96, p = .01). According to the number of variants in rs2097432 and rs393991, patients with two variants had a higher proportion of producing ATI (two variants vs. no variant, 17/21 = 80.95% vs. 9/30 = 30.00%, OR 9.92, 95% CI 2.59-37.87, p = .001; single variant vs. no variant, 30/53 = 56.60% vs. 9/30 = 30.00%, OR 3.04, 95% CI 1.18-7.88, p = .02). No association was found between other SNPs and ATI production. Conclusion: Rs2097432 in HLA-DQA1*05 and rs396991 in FCGR3A are associated with ATI production in Chinese patients with CD. A pharmacogenomic strategy could help with the clinical management of CD.

Diagnosis value of miR-181, miR-652, and CA72-4 for gastric cancer.

PURPOSE: To find a useful disease marker for early diagnosis of gastric cancer, we tried to explore the expression of serum miR-181, miR-652, and carbohydrate antigen 72-4 (CA72-4). PATIENTS AND METHODS: According to clinical pathologic stages, 112 patients with gastric cancer were divided into early gastric cancer group (n = 60) and advanced gastric cancer group (n = 52), stage I-II (n = 65), and stage III-IV (n = 47). Another 50 cases of gastric benign lesions and 40 healthy controls were also selected. Real-time quantitative PCR together with chemiluminescence were applied to detect expression levels. ROC curve was applied to judge their diagnostic efficiency. Pearson's correlation analysis was put into use to investigate the relevance of three indicators. RESULTS: Compared with benign lesions group and control group, significantly higher expression levels were found in patients of gastric cancer (all p < 0.001). Similarly, compared with early gastric cancer group, significantly higher expression levels were found in advanced gastric cancer group (all p < 0.001). The same result was also found in stage III-IV (all p < 0.001). The best cutoff values were 0.93, 2.38, and 16.94 U/ml, respectively. The area under the curve (0.917, 95%CI: 0.856-0.975) of the three combined diagnosis of early gastric cancer was the largest, and its sensitivity and specificity were 92.5% and 86.8%. And miR-181 and miR-652 were positively correlated with CA72-4 (r = 0.772, p < 0.001, r = 0.853, p < 0.001). CONCLUSION: Serum miR-181, miR-652, and CA72-4 are closely linked to the occurrence and development of gastric cancer. Combination of three indicators has diagnostic value for early gastric cancer.

Prediction of early childhood obesity with machine learning and electronic health record data.

OBJECTIVE: This study compares seven machine learning models developed to predict childhood obesity from age > 2 to ≤ 7 years using Electronic Healthcare Record (EHR) data up to age 2 years. MATERIALS AND METHODS: EHR data from of 860,510 patients with 11,194,579 healthcare encounters were obtained from the Children's Hospital of Philadelphia. After applying stringent quality control to remove implausible growth values and including only individuals with all recommended wellness visits by age 7 years, 27,203 (50.78 % male) patients remained for model development. Seven machine learning models were developed to predict obesity incidence as defined by the Centers for Disease Control and Prevention (age/sex adjusted BMI>95th percentile). Model performance was evaluated by multiple standard classifier metrics and the differences among seven models were compared using the Cochran's Q test and post-hoc pairwise testing. RESULTS: XGBoost yielded 0.81 (0.001) AUC, which outperformed all other models. It also achieved statistically significant better performance than all other models on standard classifier metrics (sensitivity fixed at 80 %): precision 30.90 % (0.22 %), F1-socre 44.60 % (0.26 %), accuracy 66.14 % (0.41 %), and specificity 63.27 % (0.41 %). DISCUSSION AND CONCLUSION: Early childhood obesity prediction models were developed from the largest cohort reported to date. Relative to prior research, our models generalize to include males and females in a single model and extend the time frame for obesity incidence prediction to 7 years of age. The presented machine learning model development workflow can be adapted to various EHR-based studies and may be valuable for developing other clinical prediction models.

Value of Bedside Lung Ultrasound in Severe and Critical COVID-19 Pneumonia.

BACKGROUND: Lung ultrasound (LUS) is an effective imaging modality that can differentiate pathological lung from non-diseased lung. We aimed to explore the value of bedside LUS in patients with severe and critical coronavirus disease 2019 (COVID-19)-associated lung injury. METHODS: Sixty-three severe and 33 critical hospitalized subjects with COVID-19 were enrolled in this study. Bedside LUS was performed in all subjects; chest computed tomography was performed on the same day as bedside LUS in 23 cases. The LUS protocol consisted of 12 scanning zones. LUS score based on B-lines and lung consolidation was evaluated. RESULTS: The most common abnormality of LUS was the various forms of B-lines, detected in 93 (96.9%) subjects; as the second most frequent abnormality, 80 (83.3%) subjects exhibited lung consolidation, mainly located in the posterior lung region. Twenty-four (25.0%) subjects had pleural line abnormalities, and 16 (16.7%) had pleural effusion; 78 (81.3%) subjects had ≥ 2 abnormal LUS patterns, and 93 (96.9%) had bilateral lung involvement. The proportion of bilateral or unilateral lung consolidation and pleural effusion in the critical COVID-19 group were higher than that in the severe group (P < .05). The lung consolidation of critical subjects showed a marked increase in most lung areas, including bilateral lateral lung, posterior lung, and left anterior-inferior lung area. The median (interquartile range) LUS scores of critical cases were higher than those of severe cases: left: 14 (12-17) vs 7 (5-12); right: 14 (10-16) vs 8 (3-12); bilateral: 28 (23-31) vs 15 (8-22) (P < .001 for all). There was a good correlation between the LUS score and the chest computed tomography score (r = 0.887, P < .001). CONCLUSIONS: The most common abnormal LUS pattern in subjects with severe and critical COVID-19 pneumonia was B-lines, followed by lung consolidation. Bedside LUS can provide important information for pulmonary involvement in patients with COVID-19.

Deep Vein Thrombosis in Hospitalized Patients With COVID-19 in Wuhan, China: Prevalence, Risk Factors, and Outcome.

BACKGROUND: To investigate deep vein thrombosis (DVT) in hospitalized patients with coronavirus disease 2019 (COVID-19), we performed a single institutional study to evaluate its prevalence, risk factors, prognosis, and potential thromboprophylaxis strategies in a large referral and treatment center. METHODS: We studied a total of 143 patients with COVID-19 from January 29, 2020 to February 29, 2020. Demographic and clinical data, laboratory data, including ultrasound scans of the lower extremities, and outcome variables were obtained, and comparisons were made between groups with and without DVT. RESULTS: Of the 143 patients hospitalized with COVID-19 (age 63±14 years, 74 [51.7%] men), 66 patients developed lower extremity DVT (46.1%: 23 [34.8%] with proximal DVT and 43 [65.2%] with distal DVT). Compared with patients who did not have DVT, patients with DVT were older and had a lower oxygenation index, a higher rate of cardiac injury, and worse prognosis, including an increased proportion of deaths (23 [34.8%] versus 9 [11.7%]; P=0.001) and a decreased proportion of patients discharged (32 [48.5%] versus 60 [77.9%]; P<0.001). Multivariant analysis showed an association only between CURB-65 (confusion status, urea, respiratory rate, and blood pressure) score 3 to 5 (odds ratio, 6.122; P=0.031), Padua prediction score ≥4 (odds ratio, 4.016; P=0.04), D-dimer >1.0 µg/mL (odds ratio, 5.818; P<0.014), and DVT in this cohort, respectively. The combination of a CURB-65 score 3 to 5, a Padua prediction score ≥4, and D-dimer >1.0 µg/mL has a sensitivity of 88.52% and a specificity of 61.43% for screening for DVT. In the subgroup of patients with a Padua prediction score ≥4 and whose ultrasound scans were performed >72 hours after admission, DVT was present in 18 (34.0%) patients in the subgroup receiving venous thromboembolism prophylaxis versus 35 (66.0%) patients in the nonprophylaxis group (P=0.010). CONCLUSIONS: The prevalence of DVT is high and is associated with adverse outcomes in hospitalized patients with COVID-19. Prophylaxis for venous thromboembolism may be protective in patients with a Padua protection score ≥4 after admission. Our data seem to suggest that COVID-19 is probably an additional risk factor for DVT in hospitalized patients.

TRIM37 targets AKT in the growth of lung cancer cells.

BACKGROUND: TRIM37 is an ubiquitin E3 ligase. Growing evidence has demonstrated the high value of TRIM37 as a potential biomarker for diagnosis of certain cancers. However, the biological function of TRIM37 in lung cancer is still unknown. MATERIALS AND METHODS: In order to gain a deep insight into the function of TRIM37 in lung cancer cells, in the present study lentiviral vector was used to mediate RNA interference and overexpression of TRIM37 in lung cancer cells (H292, H358, and H1299). In addition, a specific AKT inhibitor LY294002 was utilized to examine the correlation between the expression of TRIM37 and AKT. RESULTS: TRIM37 acts as a positive regulator of cell proliferation in lung cancer cells. Moreover, cell apoptosis analyses showed the antiapoptosis function of TRIM37, which was mainly dependent on the regulation of BCL2 and BAX. Our results also indicated that AKT might be a target of TRIM37 in lung cancer cells. CONCLUSION: This research not only helps in understanding the molecular mechanisms of TRIM37 in detail but also provides evidence to develop novel biomarkers for lung cancer diagnosis.

Association Between Left Renal Vein Entrapment and Varicocele Recurrence: A Cohort Study in 3042 Patients.

The recurrence rates after varicocelectomy vary from 0.9% to 32.2%, especially for patients with the left renal vein entrapment (LRVE). This study aims to study the association between LRVE and varicocele recurrence, and to find the risk factors of LRVE. With the design of a cohort study, we included 3042 varicocele patients who would undergo modified inguinal microscope-assisted varicocelectomy (MHMV). 858 (28.21%) patients with LRVE were as the study group, and 2184 (71.79%) patients without LRVE were as the control group. Compared with the control group, BMI was lower (p < 0.001) in study group. Totally, 18 patients had recurrence after surgery, so the recurrence rate was 0.59%. Seventeen patients (1.98%) in study group and 1 patients (0.05%) in control group had recurrence, and significant statistical difference was found between the two groups (p < 0.001). The risk ratio of LRVE for varicocele recurrence is 43.27. In conclusion, the recurrence rate of our MHMV is the lowest (0.59%). There is association between LRVE and varicocele recurrence, and varicocele patients with LRVE have higher probability of recurrence rate after varicocelectomy. BMI could be a risk factor of LRVE. Thus, for varicocele patients, especially those with lower BMI, attentions should be payed to LRVE.

Knockdown of Rhotekin 2 expression suppresses proliferation and induces apoptosis in colon cancer cells.

Colon cancer is one of the most common malignant tumors in the human body, ranking second as a gastrointestinal tumor. It has a high incidence in Europe, America and China and more than 1 million new cases of colon cancer are reported worldwide each year. The incidence of colon cancer in China has increased from 12/0.1 million in the early 1970s to 56/0.1 million at present with an annual growth rate of 4.2%, which far exceeds the international level (2%). Rhotekin (RTKN) 2, a Rho-guanosine triphosphatase (GTPase) effector, has been reported to be anti-apoptotic. However, the molecular mechanism underlying the biological function of RTKN2 in colon cancer remains unknown. The present study investigated whether the mRNA expression level of RTKN2 was markedly higher in 30 human colon cancer specimens compared with adjacent non-cancerous tissues. The results showed that the protein expression level of RTKN2 was significantly higher in SW480 and HCT116 cells, compared with HIEC cells. Knockdown of RTKN2 in the SW480 and HCT116 colon cancer cells, by lentivirus-mediated RNA interference led to the notable inhibition of cell proliferation and cell cycle progression, by reducing the expression levels of the PCDA, Cyclin D1 and c-myc cell cycle-associated proteins. The inhibitory effect of RTKN2 silencing on the proliferation of colon cancer cells may be partially realized by inhibiting the Wnt/ß-catenin signaling pathway. Furthermore, the silencing of RTKN2 in the cells induced apoptosis by reducing the expression level of Bax and increasing the expression level of Bcl2. These results show that RTKN2 is involved in the carcinogenesis and progression of human colon cancer, indicating that RTKN2 may be a molecular target in colon cancer therapy.

Expression of CD40/CD40L in colon cancer, and its effect on proliferation and apoptosis of SW48 colon cancer cells.

PURPOSE: To study was to study the expression of CD40/CD40L in colon cancer and investigate the effects of CD40/CD40L overexpression on the proliferation and apoptosis of SW48 colon cancer cell line. METHODS: Immunohistochemistry was used to detect the expression of CD40 protein in colon cancer tissue samples from 70 patients, and 10 adjacent normal tissue samples. A CD40L gene-containing plasmid was transfected into SW48 colon cancer cells using lipofectamine 2000. Cell proliferation was measured by MTT assay. The expression of CD40/CD40L was measured by real-time PCR (RT-PCR). Protein expression of Bcl-2 and Bax was detected by Western blot. RESULTS: Immunohistochemical analysis showed that CD40 was mainly expressed in the cell membrane and scarcely in the cytoplasm. The expression of CD40 in colorectal cancer tissue was significantly higher than in normal adjacent tissue. RT-PCR showed that CD40 was highly expressed in SW48 cells. The expression of CD40L mRNA was significantly increased in SW40 cells transfected with the CD40L gene-containing plasmid (p<0.01). MTT assay showed that the activity of CD40L transfected cells was significantly inhibited compared with control cells transfected with empty plasmid (p<0.01). Western blot analysis demonstrated significantly decreased Bcl-2 expression, and significantly increased Bax expression in cells transfected with the CD40L gene-containing plasmid compared with the control cells (p<0.01). CONCLUSION: In conclusion, CD40 protein expression was significantly higher in colon cancer tissue compared with normal tissue, and the apoptosis of SW48 colon cancer cells can be induced by CD40L gene transfection.

Fast and Effective Ion Mobility-Mass Spectrometry Separation of d-Amino-Acid-Containing Peptides.

Despite often minute concentrations in vivo, d-amino acid containing peptides (DAACPs) are crucial to many life processes. Standard proteomics protocols fail to detect them as d/l substitutions do not affect the peptide parent and fragment masses. The differences in fragment yields are often limited, obstructing the investigations of important but low abundance epimers in isomeric mixtures. Separation of d/l-peptides using ion mobility spectrometry (IMS) was impeded by small collision cross section differences (commonly ∼1%). Here, broad baseline separation of DAACPs with up to ∼30 residues employing trapped IMS with resolving power up to ∼340, followed by time-of-flight mass spectrometry is demonstrated. The d/l-pairs coeluting in one charge state were resolved in another, and epimers merged as protonated species were resolved upon metalation, effectively turning the charge state and cationization mode into extra separation dimensions. Linear quantification down to 0.25% proved the utility of high resolution IMS-MS for real samples with large interisomeric dynamic range. Very close relative mobilities found for DAACP pairs using traveling-wave IMS (TWIMS) with different ion sources and faster IMS separations showed the transferability of results across IMS platforms. Fragmentation of epimers can enhance their identification and further improve detection and quantification limits, and we demonstrate the advantages of online mobility separated collision-induced dissociation (CID) followed by high resolution mass spectrometry (TIMS-CID-MS) for epimer analysis.

Enhanced suppression of polyclonal CD8+25+ regulatory T cells via exosomal arming of antigen-specific peptide/MHC complexes.

Compared with CD4+25+ regulatory T cells (Tregs), the mechanisms for natural, polyclonal CD8+25+ Treg immune suppression have been significantly less studied. We previously showed that polyclonal T cells can acquire antigen-specific targeting activity through arming with exosomal peptide-MHC (pMHC). In this study, we assessed the suppressive effect of CD8+25+ Tregs or CD8+25+ Tregs armed with ovalbumin (OVA)-specific exosomes on other immune cells and OVA-specific dendritic cell (DCOVA)-stimulated antitumor immunity. We demonstrate that CD8+25+ Tregs inhibit T cell proliferation in vitro in a cell contact-dependent fashion but independent of the expression of immunosuppressive IL-10, TGF-ß, and CTLA-4. CD8+25+ Tregs anergize naïve T cells upon stimulation by up-regulating T cell anergy-associated Egr2 and down-regulating IL-2 production. Tregs also anergize DCs by preventing DC maturation through the down-regulation of Iab, CD80, CD86, and inflammatory cytokines, leading to defects in T cell stimulation. Moreover, CD8+25+ Tregs inhibit CTLs through inducing CTL death via perforin-mediated apoptosis and through reducing effector CTL cytotoxic activity via down-regulating CTL perforin-production and degranulation. In addition, we show that CD8+25+ Tregs suppress DCOVA-stimulated CTL responses in priming and effector phases and inhibit immunity against OVA-expressing CCLOVA lung cancer. Remarkably, polyclonal CD8+25+ Tregs armed with OVA-specific exosomal pMHC class-II (pMHC-II), or pMHC class-I (pMHC-I) complexes exert their enhanced inhibition of CTL responses in the priming and the effector phases, respectively. Taken together, our investigation reveals that assigning antigen specificity to nonspecific polyclonal CD8+25+ Tregs for enhanced immune suppression can be achieved through exosomal pMHC arming. This principle may have a great effect on Treg-mediated immunotherapy of autoimmune diseases.

Structural Characterization of Monomers and Oligomers of D-Amino Acid-Containing Peptides Using T-Wave Ion Mobility Mass Spectrometry.

The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer's disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers. Graphical Abstract ᅟ.

Multiple gas-phase conformations of proline-containing peptides: is it always cis/trans isomerization?

Ion mobility-mass spectrometry (IM-MS) is often employed to look at the secondary, tertiary, and quaternary structures of naked peptides and proteins in the gas-phase. Recently, it has offered a unique glimpse into proline-containing peptides and their cis/trans Xxx-Pro isomers. An experimental "signature" has been identified wherein a proline-containing peptide has its Pro residues substituted with another amino acid and the presence or absence of conformations in the IM-MS spectra is observed. Despite the high probability that one could attribute these conformations to cis/trans isomers, it is also possible that cis/trans isomers are not the cause of the additional conformations in proline-containing peptides. However, the experimental evidence of such a system has not been demonstrated or reported. Herein, we present the IM-MS analysis of Neuropeptide Y's wild-type (WT) signal sequence and Leu7Pro (L7P) mutant. Although comparison of arrival times and collision cross-sections of [M + 4H](4+) ions yields the cis/trans "signature", molecular dynamics indicates that a cis-Pro7 is not very stable and that trans-Pro7 conformations of the same cross-section arise with equal frequency. We believe that this work further underscores the importance of theoretical calculations in IM-MS structural assignments.

Multi-state targeting machinery govern the fidelity and efficiency of protein localization.

Proper localization of newly synthesized proteins is essential to cellular function. Among different protein localization modes, the signal recognition particle (SRP) and SRP receptor (SR) constitute the conserved targeting machinery in all three life kingdoms and mediate about one third of the protein targeting reactions. Based on experimental and computational studies, a detailed molecular model is proposed to explain how this molecular machinery governs the efficiency and fidelity of protein localizations. In this targeting machinery, two distinct SRP GTPases are contained into the SRP and SR that are responsible to the interactions between SRP and SR. These two GTPases can interact with one another through a series of sequential and discrete interaction states that are the early intermediate formation, stable complex association, and GTPase activation. In contrast to canonical GTPases, a floppy and open conformation adopted in free SRP GTPases can facilitate efficient GTP/GDP exchange without the aid of any external factors. As the apo-form free SRP GTPases can adopt the conformational states of GDP- or GTP-bound form, the binding of GTP/GDP follows a mechanism of conformational selection. In the first step of complex formation, the two SRP GTPases can rapidly assemble into an unstable early intermediate by selecting and stabilizing one another's primed states from the equilibrium conformational ensemble. Subsequently, extensive inter- and intra-domain changes rearrange the early complex into a tight and closed state of stable complex through induced fit mechanism. Upon stable complex association, further tune of several important interaction networks activates the SRP GTPase for GTP hydrolysis. These different conformational states are coupled to corresponding protein targeting events, in which the complex formation deliveries the translating ribosome to the target membrane and the GTPase activation couples to the cargo release from SRP-SR machinery to the translocation channel. It is thus suggested that the SRP GTPases constitute a self-sufficient system to execute exquisite spatial and temporal control of the complex targeting process. The working mechanism of the SRP and SR provides a novel paradigm of how the protein machinery functions in controlling diverse biological processes efficiently and faithfully.

A simple but effective modeling strategy for structural properties of non-heme Fe(II) sites in proteins: test of force field models and application to proteins in the AlkB family.

To facilitate computational study of proteins in the AlkB family and related α-ketoglutarate/Fe(II)-dependent dioxygenases, we have tested a simple modeling strategy for the non-heme Fe(II) site in which the iron is represented by a simple +2 point charge with Lennard-Jones parameters. Calculations for an AlkB active site model in the gas phase and ∼150 ns molecular dynamics (MD) simulations for two enzyme-dsDNA complexes (E. coli AlkB-dsDNA and ABH2-dsDNA) suggest that this simple modeling strategy provides a satisfactory description of structural properties of the Fe(II) site in AlkB enzymes, provided that care is exercised to control the binding mode of carboxylate (Asp) to the iron. MD simulations using the model for AlkB-dsDNA and ABH2-dsDNA systems find that although the structural features for the latter are overall in good agreement with the crystal structure, the dsDNA, and AlkB-dsDNA interface undergo substantial changes during the MD simulations from the crystal structure. Even for ABH2, new interactions form between a long loop region and dsDNA upon structural relaxation of the loop, supporting the role of this loop in DNA binding despite the lack of interactions between them in the crystal structure. Analysis of DNA backbone torsional distributions helps identify regions that adopt strained conformations. Collectively, the results highlight that crystal packing may have a significant impact on the structure of protein-DNA complexes; the simulations also provide additional insights regarding why AlkB and ABH2 prefer single-strand and double-strand DNA, respectively, as substrate.

FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA.

N(6)-methyladenosine is a prevalent internal modification in messenger RNA and non-coding RNA affecting various cellular pathways. Here we report the discovery of two additional modifications, N(6)-hydroxymethyladenosine (hm(6)A) and N(6)-formyladenosine (f(6)A), in mammalian messenger RNA. We show that Fe(II)- and α-ketoglutarate-dependent fat mass and obesity-associated (FTO) protein oxidize N(6)-methyladenosine to generate N(6)-hydroxymethyladenosine as an intermediate modification, and N(6)-formyladenosine as a further oxidized product. N(6)-hydroxymethyladenosine and N(6)-formyladenosine have half-life times of ~3 h in aqueous solution under physiological relevant conditions, and are present in isolated messenger RNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent N(6)-methyladenosine in messenger RNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.

Screening and test of CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector.

This study is designed to screen the CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector. Analysis results showed 414 differential genes expression, including upregulation of 209 genes and downregulation of 205 genes. Basing on the ratio of signal in experimental group to signal in control group, 45 genes (38 genes upregulation and seven genes downregulation) with significant (P<0.01) change in expression levels were screened according to the screening standard (signal log ratio≥1 or ≤-1). These genes involved into metabolism, cell cycle and apoptosis, signal transduction and stress response. Furthermore, PI3K mRNA expression level in PI3K siRNA transfected AGS cells was 0.2335±0.0116 72 h after transfection. This value was significantly (P<0.05) lower than that in blank and negative control groups. PI3K protein expression in PI3K siRNA transfected AGS cells was significantly (P<0.05) lower than that in blank and PI3K siRNA/N transfected groups. Therefore, PI3K siRNA gene silencing vector can significantly inhibit PI3K mRNA and protein expression in AGS cells.

Antagonist binding and induced conformational dynamics of GPCR A2A adenosine receptor.

The A2A adenosine receptor (A2AAR) is a unique G-protein coupled receptor (GPCR), because besides agonist, its antagonist could also lead to therapeutic relevance. Based on A2AAR-antagonist crystal structure, we have studied the binding mechanism of two distinct antagonists, ZM241385 and KW6002, and dynamic behaviors of A2AAR induced by antagonist binding. Key residues interacting with both antagonists and residues specifically binding to one of them are identified. ZM241385 specifically bound to S67(2.65), M177(5.38), and N253(6.55), while KW6002 binds to F62(2.60), A81(3.29), and H264(7.29). Moreover, interactions with L167(5.28) are found for both antagonists, which were not reported in agonist binding. The dynamic behaviors of antagonist bound holo-A2AARs were found to be different from the apo-A2AAR in three typical functional switches, (i) the "ionic lock" was in equilibrium between formation and breakage in apo-A2AAR, but stayed broken in holo-A2AARs; (ii) the "rotamer toggle switch," T88(3.36)/F242(6.44)/W246(6.48), adopted different rotameric conformations in apo-A2AAR and holo-A2AARs; (iii) apo-A2AAR preferred α-helical intracellular loop (IC)2 and flexible IC3, while holo-A2AARs had a flexible IC2 and α-helical IC3. Our results indicated that antagonist binding induced different conformational rearrangements of these characteristic functional switches in apo-A2AAR and holo-A2AARs.