[Intestinal and pharyngeal microbiota in early neonates: an analysis based on high-throughput sequencing]. / åŸºäºŽé«˜é€šé‡æµ‹åºåˆ†æžæ—©æœŸæ–°ç”Ÿå„¿è‚ é“å’Œå’½éƒ¨å¾®ç”Ÿç‰©èŒç¾¤.

OBJECTIVES: To investigate the distribution characteristics and correlation of intestinal and pharyngeal microbiota in early neonates. METHODS: Full-term healthy neonates who were born in Shanghai Pudong New Area Maternal and Child Health Hospital from September 2021 to January 2022 and were given mixed feeding were enrolled. The 16S rRNA sequencing technique was used to analyze the stool and pharyngeal swab samples collected on the day of birth and days 5-7 after birth, and the composition and function of intestinal and pharyngeal microbiota were analyzed and compared. RESULTS: The diversity analysis showed that the diversity of pharyngeal microbiota was higher than that of intestinal microbiota in early neonates, but the difference was not statistically significant (P>0.05). On the day of birth, the relative abundance of Proteobacteria in the intestine was significantly higher than that in the pharynx (P<0.05). On days 5-7 after birth, the relative abundance of Actinobacteria and Proteobacteria in the intestine was significantly higher than that in the pharynx (P<0.05), and the relative abundance of Firmicutes in the intestine was significantly lower than that in the pharynx (P<0.05). At the genus level, there was no significant difference in the composition of dominant bacteria between the intestine and the pharynx on the day of birth (P>0.05), while on days 5-7 after birth, there were significant differences in the symbiotic bacteria of Streptococcus, Staphylococcus, Rothia, Bifidobacterium, and Escherichia-Shigella between the intestine and the pharynx (P<0.05). The analysis based on the database of Clusters of Orthologous Groups of proteins showed that pharyngeal microbiota was more concentrated on chromatin structure and dynamics and cytoskeleton, while intestinal microbiota was more abundant in RNA processing and modification, energy production and conversion, amino acid transport and metabolism, carbohydrate transport and metabolism, coenzyme transport and metabolism, and others (P<0.05). The Kyoto Encyclopedia of Genes and Genomes analysis showed that compared with pharyngeal microbiota, intestinal microbiota was more predictive of cell motility, cellular processes and signal transduction, endocrine system, excretory system, immune system, metabolic diseases, nervous system, and transcription parameters (P<0.05). CONCLUSIONS: The composition and diversity of intestinal and pharyngeal microbiota of neonates are not significantly different at birth. The microbiota of these two ecological niches begin to differentiate and gradually exhibit distinct functions over time.

Metabolic activity via 18F-FDG PET/CT is predictive of microsatellite instability status in colorectal cancer.

PURPOSE: Identification of microsatellite instability high (MSI-H) colorectal cancer (CRC) is crucial for screening patients most likely to benefit from immunotherapy. We aim to investigate whether the metabolic characteristics is related to MSI status and can be used to predict the MSI-H CRC. METHODS: A retrospective analysis was conducted on 420 CRC patients who were identified via [18F]fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography(CT) prior to therapy. Maximum standardized uptake (SUVmax), mean standardized uptake (SUVmean), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) of the primary tumor were calculated and compared between MSI-H and microsatellite stability (MSS). Predictive factors of MSI status were selected from metabolic parameters and clinicopathological profiles via a multivariate analysis. RESULTS: Of 420 colorectal cancers, 44 exhibited a high incidence of MSI. Both MTV and TLG were significantly higher in MSI-H group compared with the MSS group (P = 0.004 and P = 0.010, respectively). Logistic regression analysis indicated that CRC with MSI-H were related to younger age (P = 0.013), primary lesion located at right hemi-colon (P < 0.001) and larger MTV on PET/CT imaging (P = 0.019). MTV more than 32.19 of colorectal cancer was linked to the presence of MSI (P = 0.019). CONCLUSION: Tumor metabolic burden were higher in MSI-H CRC which may be useful for predicting the MSI status of CRC patient and thus aid in determination of immunotherapy for patients with CRC.

WDR63 inhibits Arp2/3-dependent actin polymerization and mediates the function of p53 in suppressing metastasis.

Accumulating evidence suggests that p53 plays a suppressive role in cancer metastasis, yet the underlying mechanism remains largely unclear. Regulation of actin dynamics is essential for the control of cell migration, which is an important step in metastasis. The Arp2/3 complex is a major nucleation factor to initiate branched actin polymerization that drives cell migration. However, it is unknown whether p53 could suppress metastasis through modulating Arp2/3 function. Here, we report that WDR63 is transcriptionally upregulated by p53. We show with migration assays and mouse xenograft models that WDR63 negatively regulates cell migration, invasion, and metastasis downstream of p53. Mechanistically, WDR63 interacts with the Arp2/3 complex and inhibits Arp2/3-mediated actin polymerization. Furthermore, WDR63 overexpression is sufficient to dampen the increase in cell migration, invasion, and metastasis induced by p53 depletion. Together, these findings suggest that WDR63 is an important player in the regulation of Arp2/3 function and also implicate WDR63 as a critical mediator of p53 in suppressing metastasis.

Regulating Twisted Skeleton to Construct Organ-Specific Perylene for Intensive Cancer Chemotherapy.

The systemic use of pharmaceutical drugs for cancer patients is a compromise between desirable therapy and side effects because of the intrinsic shortage of organ-specific pharmaceutical drug. Design and construction of pharmaceutical drug to achieve the organ-specific delivery is thus desperately desirable. We herein regulate perylene skeleton to effect organ-specificity and present an example of lung-specific distribution on the basis of bay-twisted PDIC-NC. We further demonstrate that PDIC-NC can target into mitochondria to act as cellular respiration inhibitor, inducing insufficient production of adenosine triphosphate, promoting endogenous H2 O2 and . OH burst, elevating calcium overload, efficiently triggering the synergistic apoptosis, autophagy and endoplasmic reticulum stress of lung cancer cells. The antitumor performance of PDIC-NC is verified on in vivo xenografted, metastasis and orthotopic lung cancer, presenting overwhelming evidences for potentially clinical application. This study contributes a proof-of-concept demonstration of twisted perylene to well attain lung-specific distribution, and meanwhile achieves intensive lung cancer chemotherapy.

Inhibition of the proliferation, migration, and invasion of human breast cancer cells by leucine aminopeptidase 3 inhibitors derived from natural marine products.

Leucine aminopeptidase 3 is involved in the progression and metastasis of several cancers. This study aimed to screen anti-tumor lead compounds targeting leucine aminopeptidase 3. The compounds' suppression effect on enzyme activity and anti-tumor activity were evaluated through a series of assays. Leucine aminopeptidase 3 overexpression K562 cells were used as an enzyme source to screen 43 natural marine compounds. Compounds 5 and 6 exhibited high suppression effect on leucine aminopeptidase 3 activity. Cell activity tests indicated that both compounds have an anti-proliferative effect on triple-negative breast cancer cells. Wound healing assay and transwell invasion assay showed that both compounds could inhibit the migration and invasion of breast cancer cells. Immunoblot analysis exhibited that both compounds could downregulate the expression of metastasis-related proteins fascin and matrix metalloproteinase-2/9. A molecular dynamic simulation process was applied to discover the key features of compounds 5 and 6 in binding to leucine aminopeptidase 3 active site. This study described the anti-tumor effects of two leucine aminopeptidase 3 small molecule inhibitors. Taken together, compounds 5 and 6 could be used as anti-tumor lead compounds targeting leucine aminopeptidase 3.

Gastric cancer and image-derived quantitative parameters: Part 2-a critical review of DCE-MRI and 18F-FDG PET/CT findings.

There is yet no consensus on the application of functional imaging and qualitative image interpretation in the management of gastric cancer. In this second part, we will discuss the role of image-derived quantitative parameters from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) in gastric cancer, as both techniques have been shown to be promising and useful tools in the clinical decision making of this disease. We will focus on different aspects including aggressiveness assessment, staging and Lauren type discrimination, prognosis prediction and response evaluation. Although both the number of articles and the patients enrolled in the studies were rather small, there is evidence that quantitative parameters from DCE-MRI such as Ktrans, Ve, Kep and AUC could be promising image-derived surrogate parameters for the management of gastric cancer. Data from 18F-FDG PET/CT studies showed that standardised uptake value (SUV) is significantly associated with the aggressiveness, treatment response and prognosis of this disease. Along with the results from diffusion-weighted MRI and contrast-enhanced multidetector computed tomography presented in Part 1 of this critical review, there are additional image-derived quantitative parameters from DCE-MRI and 18F-FDG PET/CT that hold promise as effective tools in the diagnostic pathway of gastric cancer. KEY POINTS: • Quantitative analysis from DCE-MRI and18F-FDG PET/CT allows the extrapolation of multiple image-derived parameters. • Data from DCE-MRI (Ktrans, Ve, Kep and AUC) and 18F-FDG PET/CT (SUV) are non-invasive, quantitative image-derived parameters that hold promise in the evaluation of the aggressiveness, treatment response and prognosis of gastric cancer.

[Effects of wearing a mask on oxygenation of subjects with spontaneous breathing during supplementary oxygen through facemask].

During the COVID-19 epidemic, our national guidelines have suggested that surgical patients should wear a mask to decrease the potential transmission of COVID-19 in the operating room, as long as the condition allows. However, so far, there is no study to discuss the influence of wearing a mask on the ventilation and blood oxygenation status in patients of spontaneous breathing with supplementary oxygen through an anesthetic facemask. This is a before-after study in the same patient, and 10 healthy volunteers were recruited, by testing the arterial blood gas parameters at key time points before and after oxygen inhalation to evaluate the effects of two different supplementary oxygen methods ('disposable medical mask + anesthetic facemask' and 'anesthetic facemask only') on the oxygenation of subjects. Our data demonstrated whether wearing a disposable medical mask or not could effectively increase the oxygen supply of the subjects compared with the basic value before oxygen inhalation; however, compared with the group without mask, the arterial oxygen partial (PaO 2) reduced significantly at each time points when subjects wearing a disposable medical mask. There was no significant difference in other parameters, and our data showed that age growth and smoking had no significant effects on the difference of PaO 2 between the groups with and without masks. This study demonstrates effective oxygen supplementation through anesthetic facemask in subjects with spontaneous breathing who is wearing a disposable medical mask, whose pulse oxygen saturation and arterial oxygen saturation can reach 100% rapidly, and this provides a theoretical basis for the management of patients with disseminated respiratory diseases to wear masks in the operating room; however, the rate and amount of PaO 2 increase are both decreased as compared to those who is not wearing a disposable medical mask during supplementary oxygenation. Whether this difference will affect the clinical outcome needs further study.

Preparation of endogenous TopBP1/Dpb11 and effect on central checkpoint kinase Mec1- Ddc2 (human ATR-ATRIP homolog).

The Saccharomyces cerevisiae Mec1 kinase, the mammalian ATR ortholog, is essential for sensing a variety of DNA lesions and initiating DNA damage response. The Dpb11, a homolog of human TopBP1, functions in activating the Mec1 upon DNA replication stress and DNA damages. Here, we report an affinity purification and ion exchange chromatography method to efficiently purify endogenous Dpb11 under normal expression level directly from yeast whole cell extraction. The final concentration of 5 µM of high purity and homogeneity biochemical preparation enables functional and structural characterization of the physical interaction between Dpb11 and Mec1-Ddc2 complex. The Dpb11 obtained by endogenous purification strongly stimulates the Mec1 kinase activity and promotes the changes in conformational distribution. This observation suggests the Dpb11 activates Mec1 kinase probably through modulation in the kinase conformations.

New Method for Detecting the Suppressing Effect of Enzyme Activity by Aminopeptidase N Inhibitor.

Aminopeptidase N, also known as CD13, is a transmembrance protease with many functions. CD13 is involved in inflammatory diseases and cancers. A convenient and reliable laboratory test method for detecting the suppressing effects of enzyme activity would be useful for study of CD13 inhibitors. Porcine CD13 (pCD13) was traditionally considered an enzyme source but has significant practical disadvantages. pCD13 is not a human source, and the accuracy and reliability of experimental results are greatly reduced. In this study, a modified detection method with K562-CD13 monoclonal cells, a human-derived cell line, was established to detect the suppressing effects of enzyme activity by the CD13 inhibitor. In this method, K562-CD13 monoclonal cells were used as enzyme source and L-leucine p-nitroaniline hydrochloride as substrate. Using CD13 enzyme activity analyses, we found that the ability of the catalytic substrate was weaker in K562 cells than in the other cell lines, and K562-CD13 cells expressed significantly higher levels of CD13 enzyme activity than parental K562 cells. The enzyme activity of CD13 was detected with the new method after ubenimex treatment. The enzyme activity was significantly inhibited by ubenimex in a dose-dependent manner. In summary, this study proposes a sensitive, stable, and objective laboratory method for detecting the inhibitory effect of the CD13 inhibitor.

Bone marrow mesenchymal stem cells overexpressing GATA-4 improve cardiac function following myocardial infarction.

INTRODUCTION: The present study aimed to examine whether GATA-4 overexpressing bone marrow mesenchymal stem cells can improve cardiac function in a murine myocardial infarction model compared with bone marrow mesenchymal stem cells alone. METHODS: A lentiviral-based transgenic system was used to generate bone mesenchymal stem cells which stably expressed GATA-4 (GATA-4-bone marrow mesenchymal stem cells). Apoptosis and the myogenic phenotype of the bone marrow mesenchymal stem cells were measured using Western blot and immunofluorescence assays co-cultured with cardiomyocytes. Cardiac function, bone marrow mesenchymal stem cell homing, cardiac cell apoptosis, and vessel number following transplantation were assessed, as well as the expression of c-Kit. RESULTS: In GATA-4-bone marrow mesenchymal stem cells-cardiomyocyte co-cultures, expression of myocardial-specific antigens, cTnT, connexin-43, desmin, and α-actin was increased compared with bone marrow mesenchymal stem cells alone. Caspase 8 and cytochrome C expression was lower, and the apoptotic rate was significantly lower in GATA-4 bone marrow mesenchymal stem cells. Cardiac function following myocardial infarction was also increased in the GATA-4 bone marrow mesenchymal stem cell group as demonstrated by enhanced ejection fraction and left ventricular fractional shortening. Analysis of the cardiac tissue revealed that the GATA-4 bone marrow mesenchymal stem cell group had a greater number of DiR-positive cells suggestive of increased homing and/or survival. Transplantation with GATA-4-bone marrow mesenchymal stem cells significantly increased the number of blood vessels, decreased the proportion of apoptotic cells, and increased the mean number of cardiac c-kit-positive cells. CONCLUSION: GATA-4 overexpression in bone marrow mesenchymal stem cells exerts anti-apoptotic effects by targeting cytochrome C and Fas pathways, promotes the aggregation of bone marrow mesenchymal stem cells in cardiac tissue, facilitates angiogenesis, and effectively mobilizes c-kit-positive cells following myocardial infarction, leading to the improvement of cardiac function after MI.

A 18FDG PET/CT-based volume parameter is a predictor of overall survival in patients with local advanced gastric cancer.

OBJECTIVE: The present study investigated the prognosis value of preoperative fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) in patients with local advanced gastric cancer (LAGC). METHODS: In total, 144 patients [median age 63 (range: 48-80) years old] with LAGC underwent18F-FDG PET/CT prior to any treatment. The maximum standardized uptake values (SUVmax), mean standardized uptake values (SUVmean), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) of the primary lesion were measured on PET/CT and correlated with clinicopathological features and survival. RESULTS: Significant differences in SUVmean, SUVmax, MTV and TLG were found according to Lauren's classification, histologic grade and T category (P<0.05). During the 26.5-month follow-up, 51 (35.4%) patients died and 70 (48.6%) exhibited disease progression. The optimal thresholds of MTV and TLG were 15.1 cm3 and 47.3 cm3, respectively. The 3-year progression-free survival (PFS) and overall survival (OS) for patients with high TLG values were 30% and 38% compared to 38% and 47% for low TLG values, respectively (P<0.05). Univariate and multifactor analyses demonstrated that lymph node metastasis and T stage were independent prognostic factors for PFS; T stage, histologic grade and TLG were independent prognostic factors for OS (P<0.05). Molecular markers had no relationship with patient's outcomes. CONCLUSIONS: Metabolic activity of primary gastric tumors from 18F-FDG PET/CT is a prognostic factor in patients with LAGC.

Prognostic value of 18F-fluorodeoxyglucose positron emission tomography using Deauville criteria in diffuse large B cell lymphoma treated with autologous hematopoietic stem cell transplantation.

OBJECTIVE: High-dose chemotherapy (HDC) followed by autologous hematopoietic stem cell transplantation (auto-HSCT) plays an important role in improving outcomes of diffuse large B cell lymphoma (DLBCL) patients. 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography (CT) has been widely accepted in response assessment and prediction of prognosis in DLBCL. Here, we report the value of 18F-FDG PET/CT pre- and post-HSCT in predicting outcomes of patients with DLBCL. METHODS: DLBCL patients who had PET/CT scan before and after HSCT were included. PET results were interpreted based upon Deauville criteria. The prognostic value of 18F-FDG PET/CT in auto-HSCT was evaluated. RESULTS: Eighty-four patients were enrolled. In univariate analysis, pre- and post-HSCT PET findings were correlated with 3-year progression-free survival (PFS) [hazard ratio (HR)=4.391, P=0.001; HR=7.607, P<0.001] and overall survival (OS) (HR=4.792, P=0.008; HR=26.138, P<0.001). Patients receiving upfront auto-HSCT after first-line treatment had better outcomes than relapsed/refractory DLBCL patients (3-year PFS, P<0.001; 3-year OS, P<0.001). In the relapsed/refractory patients, pre- and post-HSCT PET findings were also associated with 3-year PFS (P=0.003vs. P<0.001) and OS (P=0.027vs. P<0.001). A significant correlation was observed between clinical response to chemotherapy before auto-HSCT and outcomes of patients in the entire cohort (3-year PFS, P<0.001; 3-year OS, P<0.001) and in the subgroup of 21 patients with positive pre-HSCT PET (3-year PFS, P=0.084; 3-year OS, P=0.240). A significant association between survival and post-HSCT PET findings was observed in multivariate analysis (HR=5.168, P<0.001). CONCLUSIONS: PET results before and after HSCT are useful prognostic factors for DLBCL patients receiving HSCT. Patients who responded to chemotherapy, even those with positive pre-HSCT PET, are appropriate candidates for auto-HSCT.

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.

Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a ß-prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore-forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish-specific defense molecule.

Tf2O-Promoted Intramolecular Schmidt Reaction of the ω-Azido Carboxylic Acids.

A designed Tf2O-promoted intramolecular Schmidt reaction of 2-substituted ω-azido carboxylic acids was demonstrated. Tf2O was used as an activation reagent for the carboxylic acid, and ω-azido anhydride was in situ generated, releasing a molecular TfOH, which acted as an acid promoter for the Schmidt process. A series of 2-substituted pyrrolidines was produced and acetylated for better purification. The strategy was also efficient for conversion of a 4-substituted ω-azido carboxylic acid to the tricyclic lactam.

Long-Term Effects of Bone Morphogenetic Protein-2-Loaded Calcium Phosphate on Maxillary Sinus Lift Surgery for Delayed and Simultaneous Dental Implantation.

Maxillary sinus lift surgery (MSLS) is considered to be a useful treatment method for patients with atrophic alveolar bone. Choosing a suitable surgical procedure to simultaneously decrease graft absorption and obtain long-term survival of dental implants is still a challenge. In this study, 20 patients received MSLS with graft of bone morphogenetic protein 2 (BMP2)-loaded calcium phosphate. Dental implants were placed simultaneously in 10 patients receiving MSLS (1-stage group), and in 10 patients receiving dental implants with a 3 to 6 months delay (2-stage group). The effects were evaluated based on clinical and radiographic examination during a 4 to 5 years follow-up. The results showed that only 1 perforation of the maxillary sinus mucosa was observed in 1-stage group, and was patched with a collagen membrane. An average bone gain of 6.8 mm was observed, and all implants exhibited no looseness, peri-implantitis, or fracture, all of which were stable during the follow-up and exhibited nice dental function during a 4 to 5 years follow-up. The loss of peri-implant bone height was 1.12 ±â€Š0.47 and 1.10 ±â€Š0.39 mm, the probing depth of periodontal pocket was 1.79 ±â€Š0.62 and 1.81 ±â€Š0.71 mm, the sulcular bleeding index was 1.63 ±â€Š0.47 and 1.72 ±â€Š0.54 in 1-stage group and 2-stage group, respectively, and no significant differences existed between these 2 groups. These findings implied that BMP2-loaded calcium phosphate may be a suitable material for MSLS, especially for patients with minimal bone height. Clinicians can use the 1- or 2-stage technique based on clinical condition, patients' choice and clinicians' experience. In patients where implants cannot be stabilized for patients with minimal bone height, 2-stage surgery may be more suitable.

Prognostic value of pre- and post-transplantation 18F-fluorodeoxyglucose positron emission tomography results in non-Hodgkin lymphoma patients receiving autologous stem cell transplantation.

OBJECTIVE: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) is the standard of care in the upfront or relapsed/refractory setting in some patients with non-Hodgkin lymphoma (NHL). However, a proportion of patients do not respond to ASCT. 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) has been widely used for staging, response evaluation, and prognosis prediction. Here, we investigated the prognostic role of PET/CT in NHL patients before and after ASCT. METHODS: A retrospective study was conducted at Peking University Cancer Hospital. All NHL patients who underwent ASCT between March 2010 and July 2016 were identified. Patients who had PET/CT scan before and after ASCT were included. Deauville criteria (5-point scale) were used to interpret PET scans. Univariate and multivariate survival analyses were performed using Cox regression. The predictive value of PET scanning was estimated by comparing the area under the receiver operating characteristic (ROC) curve. RESULTS: In total, 79 patients were enrolled in this study. In univariate analysis, pre- and post-ASCT PET result was identified as prognostic factors for 3-year progression-free survival (PFS) and overall survival (OS). Patients with negative pre-ASCT PET result demonstrated significantly better PFS (84.2% vs. 54.2%) and OS (89.2% vs. 63.6%) than patients with positive pre-ASCT PET result. PFS (91.6% vs. 25.3%) and OS (96.5% vs. 36.8%) were also significantly different between patients with negative and positive post-ASCT PET result. Multivariate analysis also showed a significant association between survival and post-ASCT PET result. ROC analysis revealed that the predictive value of post-ASCT PET result was superior to that of pre-ASCT PET result alone. Combined pre- and post-ASCT PET result is better for predicting outcomes in patients with NHL receiving transplantation. Deauville criteria score >3 was identified as the best cutoff value for post-ASCT PET. CONCLUSIONS: Post-ASCT PET result was more important than pre-ASCT PET result in predicting outcomes for NHL patients who underwent ASCT. The prognostic significance can be improved when combining pre-ASCT PET result with post-ASCT PET result. Deauville criteria can be used for interpreting PET scans in this scenario.

Crystal structure of DnaT84-153-dT10 ssDNA complex reveals a novel single-stranded DNA binding mode.

DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriA-dependent primosome assembly is still a black box. Here, we report the 2.83 Å DnaT(84-153)-dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA.

Influence of chelator and near-infrared dye labeling on biocharacteristics of dual-labeled trastuzumab-based imaging agents.

OBJECTIVE: To investigate the effect of fluorescent dye labeling on the targeting capabilities of 111In- (DTPA)n-trastuzumab-(IRDye 800)m. METHODS: Trastuzumab-based conjugates were synthesized and conjugated with diethylenetriaminepentaacetic acid (DTPA) at molar ratios of 1, 2, 3 and 5 and with a fluorescent dye (IRDye 800CW) at molar ratios of 1, 3 and 5. Immunoreactivity and internalization were assessed on SKBR-3 cells, overexpressing human epidermal growth factor receptor 2. The stability in human serum and phosphate-buffered saline (PBS) was evaluated. The biodistribution of dual-labeled conjugates was compared with that of 111In-(DTPA)2-trastuzumab in a SKBR-3 xenograft model to evaluate the effect of dye-to-protein ratio. RESULTS: All trastuzumab-based conjugates exhibited a high level of chemical and optical purity. Flow cytometry results showed that increasing dye-to-protein ratios were associated with decreased immunoreactivity. Stability studies revealed that the conjugate was stable in PBS, while in human serum, increased degradation and protein precipitation were observed with increasing dye-to-protein ratios. At 4 h, the percentages of internalization of dual-labeled conjugates normalized by dye-to-protein ratio (m) were 24.88%±2.10%, 19.99%±0.59%, and 17.47%±1.26% for "m" equal to 1, 3, and 5, respectively. A biodistribution study revealed a progressive decrease in tumor uptake with an increase in the dye-to-protein ratios. The liver, spleen and kidney showed a marked uptake with increased dye-to-protein ratios, particularly in the latter. CONCLUSIONS: With non-specific-site conjugation of the fluorescent dye with a protein based on imaging agent, the increase in dye-to-protein ratios negatively impacted the immunoreactivity and stability, indicating a reduced tumor uptake.