Miracle-dz/UniFAR-FTUs huggingface dataset:
SAMURAI (Solid-phase Assisted Mutagenesis by Uracil Restriction for Accurate Integration) for antibody affinity maturation and paratope mapping

Abstract Mutagenesis libraries are essential for combinatorial protein engineering. Despite improvements in gene synthesis and directed mutagenesis, current methodologies still have limitations regarding the synthesis of complete antibody single-chain variable fragment (scFv) genes and simultaneous diversification of all six CDRs. Here, we describe the generation of mutagenesis libraries for antibody affinity maturation using a cell-free solid-phase technique for annealing of single-strand mutagenic oligonucleotides. The procedure consists of PCR-based incorporation of uracil into a wild-type template, bead-based capture, elution of single-strand DNA, and in vitro uracil excision enzyme based degradation of the template DNA. Our approach enabled rapid (8 hours) mutagenesis and automated cloning of 50 position-specific alanine mutants for mapping of a scFv antibody paratope. We further exemplify our method by generating affinity maturation libraries with diversity introduced in critical, nonessential, or all CDR positions randomly. Assessment with Illumina deep sequencing showed less than 1% wild-type in two libraries and the ability to diversify all CDR positions simultaneously. Selections of the libraries with bacterial display and deep sequencing evaluation of the selection output showed that diversity introduced in non-essential positions allowed for a more effective enrichment of improved binders compared to the other two diversification strategies.
BODE-index, modified BODE-index and ADO-score in Chronic Obstructive Pulmonary Disease: Relationship with COPD phenotypes and CT lung density changes

Abstract COPD is a heterogeneous disorder whose assessment is going to be increasingly multidimensional. Grading systems such as BODE (Body-Mass Index, Obstruction, Dyspnea, Exercise), mBODE (BODE modified in grading of walked distance), ADO (Age, Dyspnea, Obstruction) are proposed to assess COPD severity and outcome. Computed tomography (CT) is deemed to reflect COPD lung pathologic changes. We studied the relationship of multidimensional grading systems (MGS) with clinically determined COPD phenotypes and CT lung density. Seventy-two patients underwent clinical and chest x-ray evaluation, pulmonary function tests (PFT), 6-minute walking test (6MWT) to derive: predominant COPD clinical phenotype, BODE, mBODE, ADO. Inspiratory and expiratory CT was performed to calculate mean lung attenuation (MLA), relative area with density below-950 HU at inspiration (RAI-950), and below -910 HU at expiration (RAE-910). MGS, PFT, and CT data were compared between bronchial versus emphysematous COPD phenotype. MGS were correlated with CT data. The prediction of CT density by means of MGS was investigated by direct and stepwise multivariate regression. MGS did not differ in clinically determined COPD phenotypes. BODE was more closely related and better predicted CT findings than mBODE and ADO; the better predictive model was obtained for CT expiratory data; stepwise regression models of CT data did not include 6MWT distance; the dyspnea score MRC was included only to predict RA-950 and RA-910 which quantify emphysema extent. BODE reflect COPD severity better than other MGS, but not its clinical heterogeneity. 6MWT does not significantly increase BODE predictivity of CT lung density changes.
Characteristics of Neighborhoods Where Emergency Medical Services Encounter Children at Risk for Maltreatment

Abstract Objective: The objective of this study was to determine if neighborhood rates of pediatric Emergency Medical Services (EMS) encounters correlate with rates of child maltreatment reporting and if there are neighborhood-level risk factors for EMS encountering children with maltreatment reports. Methods: We conducted a retrospective cohort study using the electronic medical records of children ages <18 years who had Columbus Division of Fire EMS encounters between 2011 and 2015. We used Nationwide Children’s Hospital electronic medical records to identify child maltreatment reports. The EMS scene addresses and home addresses associated with maltreatment reports were geocoded independently and rates for each Census tract were calculated. The maltreatment reports were matched to the EMS encounters using name, gender, and date of birth. Rates of EMS encounters with children that had a maltreatment report were calculated for each Census tract. Census tract demographic information was obtained from the American Community Survey. Bayesian conditional autoregressive Poisson models were used to calculate rate ratios for census tract variables to determine their relationship to EMS encountering children with maltreatment reports. Results: A total of 44,002 EMS encounters and 4,298 maltreatment reports were included in the study. The Spearman correlation coefficient relating rates of EMS encounters to rates of maltreatment reports within census tracts was 0.72 (95% confidence interval, 0.65–0.77). Within the study period, a total of 1,134 EMS encounters were linked to 578 children with maltreatment reports. Poverty was the only independent risk factor for EMS encountering children with maltreatment reports. The multivariate analysis also identified protective factors, which included neighborhoods with higher proportions of residents who had bachelor’s degrees, spoke a language other than English, and had the same residence the previous year. Conclusion: This study showed that in Franklin County, Ohio, neighborhoods with high EMS utilization had a strong positive correlation with areas that had high rates of child maltreatment reports. We also identified four neighborhood characteristics that were independently associated with EMS encountering children at risk for maltreatment (risk factor: poverty; protective factors: residents with college educations, non-English speaking households, and residents maintaining the same residence as the previous year).
Loss of SMAD4 Promotes Colorectal Cancer Progression by Recruiting Tumor-Associated Neutrophils via the CXCL1/8–CXCR2 Axis

Purpose: SMAD4 is a key transcriptional factor of TGFβ signaling and acts as a tumor suppressor in colorectal cancer. In the present study, we explored the immunologic effect of SMAD4 on the tumor microenvironment. Experimental Design: Using 99 clinical specimens and human colorectal cancer cell lines, we investigate the relationship between SMAD4 expression and neutrophil accumulation. We immunohistochemically analyzed expression of SMAD4, CXCL1, CXCL8, CXCR2, and other proteins with clinical specimens. Finally, we determined the serum levels of CXCL1 and CXCL8 in 125 patients with colorectal cancer. Results: SMAD4 knockdown from human colorectal cancer cells upregulated the expression of CXCL1 and CXCL8, which recruited neutrophils to colorectal cancer tumor via CXCR2. In turn, when neutrophils were exposed to the supernatant of SMAD4-negative colorectal cancer cells, they produced a large amount of CXCL1 and CXCL8 by themselves in vitro. In human clinical specimens, we found that neutrophil infiltration into the peritumoral stroma was more marked in SMAD4-negative colorectal cancer compared with that in SMAD4-positive colorectal cancer, and that both CXCL1 and CXCL8 were abundantly expressed in the tumor-infiltrating neutrophils. Neutrophils isolated from primary colorectal cancer expressed significantly higher levels of CXCL1 and CXCL8 than did those isolated from peripheral blood. Furthermore, tumor-infiltrating neutrophils expressed MMP2 and MMP9 in addition to ARG1 and IDO. Serum CXCL8 level was significantly higher in colorectal cancer patients, especially those at stage II/III, and statistical analysis indicated a high CXCL8 level was associated with a shorter overall survival and relapse-free survival. Conclusions: Blockade of the CXCL1/8–CXCR2 axis could be a novel therapeutic approach against SMAD4-negative colorectal cancer.
Cone beam computed tomography evaluation of staged lateral ridge augmentation using platelet‐rich fibrin or resorbable collagen membranes in a randomized controlled clinical trial

OBJECTIVES To evaluate the volumetric changes following lateral alveolar ridge augmentation using autogenous bone graft covered by either a platelet-rich fibrin membrane (test group) or an inorganic bovine bone substitute and a resorbable collagen barrier membrane (control group). MATERIAL AND METHODS A total of 27 partially edentulous patients (test n = 14, control n = 13) with the indication for lateral bone block augmentation were included in this randomized, controlled clinical trial. Cone beam computed tomography (CBCT) examination was performed prior to grafting and 2 weeks and 6 months after grafting. The volumetric changes between the various examinations times were evaluated by planimetric measurements on two-dimensional CBCT images of the grafted regions. RESULTS The mean bone volumetric loss in the test group was 14.7%, SD ±8.9%, while the mean bone volume loss in the control group was 17.8%, SD ±13.3%. This difference was not significant (p = 0.48). A total of ten patients were operated in the incisor and canine region with a mean bone volume loss of 23.41% SD, ±10.87%, while 17 patients were operated in the premolar region with a mean bone volume loss of 11.89% SD ±9.05%. This difference was significant (p = 0.01). CONCLUSION The test and control group demonstrated no overall difference in volumetric bone changes of the augmented bone at the 6-month follow-up. The second major finding revealed a significantly larger amount of bone resorption in the incisor and canine region than in the premolar region of the maxilla, particularly in the control group.
Immunological basis for enhanced immunity of nanoparticle vaccines

ABSTRACT Introduction: Immunization has been a remarkably successful public health intervention; however, new approaches to vaccine design are essential to counter existing and emerging infectious diseases which have defied traditional vaccination efforts to date. Nanoparticles (ordered structures with dimensions in the range of 1–1000 nm) have great potential to supplement traditional vaccines based upon pathogen subunits, or killed or attenuated microorganisms, as exemplified by the successful licensure of virus-like particle vaccines for human papillomavirus and hepatitis B. However, the immunological mechanisms that underpin the potent immunity of nanoparticle vaccines are poorly defined. Areas covered: Here, we review the immunity of nanoparticle immunization. The display of antigen in a repetitive, ordered array mimics the surface of a pathogen, as does their nanoscale size. These properties facilitate enhanced innate immune activation, improved drainage and retention in lymph nodes, stronger engagement with B cell receptors, and augmented T cell help in driving B cell activation. Expert opinion: In the near future, increasingly complex nanoparticle vaccines displaying multiple antigens and/or co-delivered adjuvants will reach clinical trials. An improved mechanistic understanding of nanoparticle vaccination will ultimately facilitate the rational design of improved vaccines for human health.
Evaluation of the anti-oxidant and anti-inflammatory effects of the methanolic extract of Ferula szowitsiana root on PHA-induced inflammation in human lymphocytes

Abstract Inflammation is defined as a defensive response of the body against either the endogenous or exogenous triggers, while this process becomes chronic leading to various disorders such as asthma, cancers, and multiple sclerosis. Recently, pharmacological properties of different constituents of F. szowitsiana have been reported. In the present study, we aimed to evaluate the anti-oxidative and anti-inflammatory effects of the methanolic extract of F. szowitsiana root on human isolated lymphocytes. The effects of either F. szowitsiana (10, 40 and 160 μg/ml) or dexamethasone (0.1 mM) were evaluated on the levels of cell proliferation, reactive oxygen species (ROS), nitric oxide (NO) production, malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activities, and total glutathione content (GSH) as well as the secretion of inflammatory cytokines [interleukin 6 (IL-6) and tumor necrosis factor (TNF)-α] in the presence or absence of phytohemagglutinin (PHA) stimulation (n = 8 for each group). PHA stimulation notably elevated ROS, NO, MDA, IL-6, and TNF-α levels as well as diminished GSH, CAT and SOD levels. In PHA-stimulated, the results also revealed that F. szowitsiana (10–160 µg/ml) significantly decreased MDA, ROS, NO, IL-6 and TNF-α levels as well as increased CAT, SOD and GSH levels. Collectively, F. szowitsiana is able to attenuate the overproduction of inflammatory and oxidative stress markers in the presence of PHA-stimulated T lymphocytes, while to propagate the anti-oxidative defense. Contextually, the plant has promising healing effects in the different inflammatory disorders associated with the interference of the acquired immune system such as multiple sclerosis and asthma.
PPI‐Detect: A support vector machine model for sequence‐based prediction of protein–protein interactions

The prediction of peptide–protein or protein–protein interactions (PPI) is a challenging task, especially if amino acid sequences are the only information available. Machine learning methods allow us to exploit the information content in PPI datasets. However, the numerical codification of these datasets often influences the performance of data mining approaches. Here, we introduce a procedure for the general‐purpose numerical codification of polypeptides. This procedure transforms pairs of amino acid sequences into a machine learning‐friendly vector, whose elements represent numerical descriptors of residues in proteins. We used this numerical encoding procedure for the development of a support vector machine model (PPI‐Detect), which allows predicting whether two proteins will interact or not. PPI‐Detect (https://ppi-detect.zmb.uni-due.de/) outperforms state of the art sequence‐based predictors of PPI. We employed PPI‐Detect for the analysis of derivatives of EPI‐X4, an endogenous peptide inhibitor of CXCR4, a G‐protein‐coupled receptor. There, we identified with high accuracy those peptides which bind better than EPI‐X4 to the receptor. Also using PPI‐Detect, we designed a novel peptide and then experimentally established its anti‐CXCR4 activity. © 2019 Wiley Periodicals, Inc.
Mutations in the DNMT3A DNA methyltransferase in acute myeloid leukemia patients cause both loss and gain of function and differential regulation by protein partners

Eukaryotic DNA methylation prevents genomic instability by regulating the expression of oncogenes and tumor-suppressor genes. The negative effects of dysregulated DNA methylation are highlighted by a strong correlation between mutations in the de novo DNA methyltransferase gene DNA methyltransferase 3α (DNMT3A) and poor prognoses among acute myeloid leukemia (AML) patients. We show here that clinically observed DNMT3A mutations dramatically alter enzymatic activity, including mutations that lead to 6-fold hypermethylation and 3-fold hypomethylation of the human cyclin-dependent kinase inhibitor 2B (CDKN2B or p15) gene promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN-AML) constitutes 40–50% of all AML cases and is the most epigenetically diverse AML subtype with pronounced changes in non-CpG DNA methylation. We identified a subset of DNMT3A mutations that enhance the enzyme's ability to perform non-CpG methylation by 2–8–fold. Many of these mutations mapped to DNMT3A regions known to interact with proteins that themselves contribute to AML, such as thymine DNA glycosylase (TDG). Using functional mapping of TDG–DNMT3A interactions, we provide evidence that TDG and DNMT3-like (DNMT3L) bind distinct regions of DNMT3A. Furthermore, DNMT3A mutations caused diverse changes in the ability of TDG and DNMT3L to affect DNMT3A function. Cell-based studies of one of these DNMT3A mutations (S714C) replicated the enzymatic studies and revealed that it causes dramatic losses of genome-wide methylation. In summary, mutations in DNMT3A lead to diverse levels of activity, interactions with epigenetic machinery components and cellular changes.
Effects of Synaptic Synchrony on the Neuronal Input-Output Relationship

The firing rate of individual neurons depends on the firing frequency of their distributed synaptic inputs, with linear and nonlinear relations subserving different computational functions. This letter explores the relationship between the degree of synchrony among excitatory synapses and the linearity of the response using detailed compartmental models of cortical pyramidal cells. Synchronous input resulted in a linear input-output relationship, while asynchronous stimulation yielded sub- and supraproportional outputs at low and high frequencies, respectively. The dependence of input-output linearity on synchrony was sigmoidal and considerably robust with respect to dendritic location, stimulus irregularity, and alteration of active and synaptic properties. Moreover, synchrony affected firing rate differently at lower and higher input frequencies. A reduced integrate-and-fire model suggested a mechanism explaining these results based on spatiotemporal integration, with fundamental implications relating synchrony to memory encoding.
Distribution of cerebral microbleeds in the East and West

Objective We investigated differences in the anatomical distribution of cerebral microbleeds (CMBs) on MRI, hypothesized to indicate the type of underlying cerebral small vessel disease (SVD), between Eastern and Western general populations. Methods We analyzed data from 11 studies identified by a PubMed search between 1996 and April 2014 according to the Preferred Reporting Items for a Systematic Review and Meta-analysis of Individual Participant Data. Study quality measures indicated low or medium risk of bias. We included stroke-free participants from populations aged between 55 and 75 years, categorized by geographic location (Eastern or Western). We categorized CMB distribution (strictly lobar, deep and/or infratentorial [D/I], or mixed [i.e., CMBs located in both lobar and D/I regions]). We tested the hypothesis that Eastern and Western populations have different anatomical distributions of CMBs using multivariable mixed effects logistic regression analyses adjusted for age, sex, and hypertension and clustering by institution. Results Among 8,595 stroke-free individuals (mean age [SD] 66.7 [5.6] years; 48% male; 42% from a Western population), 624 (7.3%) had CMBs (strictly lobar in 3.1%; D/I or mixed in 4.2%). In multivariable mixed effects models, Eastern populations had higher odds of D/I or mixed CMBs (adjusted odds ratio 2.78, 95% confidence interval [CI] 1.77–4.35) compared to Western populations. Eastern populations had a higher number of D/I or mixed CMBs (adjusted prevalence ratio 2.83, 95% CI 1.27–6.31). Conclusions Eastern and Western general populations have different anatomical distributions of CMBs, suggesting differences in the spectrum of predominant underlying SVDs, with potential implications for SVD diagnosis and treatment.
Protein Phosphatase 2A B'α and B'β Protect Centromeric Cohesion during Meiosis I1

Protein phosphatase 2A regulatory subunits B'α and B'β mediate AtREC8 dephosphorylation during meiosis and are essential for male and female germ cell fertility in Arabidopsis (Arabidopsis thaliana). During meiosis, the stepwise release of sister chromatid cohesion is crucial for the equal distribution of genetic material to daughter cells, enabling generation of fertile gametophytes. However, the molecular mechanism that protects centromeric cohesion from release at meiosis I is unclear in Arabidopsis (Arabidopsis thaliana). Here, we report that the protein phosphatase 2A regulatory subunits B'α and B'β participate in the control of sister chromatid separation. The double mutant b'αβ exhibited severe male and female sterility, caused by the lack of a nucleus or presence of an abnormal nucleus in mature microspores and embryo sacs. 4′,6-Diamidino-2-phenylindole staining revealed unequal amounts of DNA in the mononuclear microspores. Transverse sections of the anthers revealed unevenly sized tetrads with or without a nucleus, suggesting a defect in meiocyte meiosis. An analysis of chromosome spreads showed that the sister chromatids separated prematurely at anaphase I in b'αβ. Immunoblotting showed that AtRECOMBINATION DEFECTIVE8 (AtREC8), a key member of the cohesin complex, was hyperphosphorylated in b'αβ anthers and pistils during meiosis but hypophosphorylated in the wild type. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays showed that B'α and B'β interact specifically with AtREC8, AtSHUGOSHIN1 (AtSGO1), AtSGO2, and PATRONUS1. Given that B'α was reported to localize to the centromere in meiotic cells, we propose that protein phosphatase 2A B'α and B'β are recruited by AtSGO1/2 and PATRONUS1 to dephosphorylate AtREC8 at the site of centromere cohesion to shield it from cleavage until anaphase II, contributing to the balanced separation of sister chromatids at meiosis.
The intestinal microbiota predisposes to traveler’s diarrhea and to the carriage of multidrug-resistant Enterobacteriaceae after traveling to tropical regions

ABSTRACT The risk of acquisition of multidrug-resistant Enterobacteriaceae (MRE) and of occurrence of diarrhea is high when traveling to tropical regions. The relationships between these phenomena and the composition of human gut microbiota have not yet been assessed. Here, we investigated the dynamics of changes of metabolically active microbiota by sequencing total RNA from fecal samples taken before and after travel to tropical regions. We included 43 subjects who could provide fecal samples before and after a travel to tropical regions. When found positive by culturing for any MRE after travel, the subjects sent an additional sample 1 month later. In all, 104 fecal samples were considered (43 before travel, 43 at return, 18 one month after travel). We extracted the whole RNA, performed retrotranscription and sequenced the cDNA (MiSeq 2x300bp). The reads were mapped to the reference operational taxonomic units (OTUs) and species/strains using the 16S Greengenes and 23S SILVA databases. We found that the occurrence of diarrhea during the travel was associated with a higher relative abundance of Prevotella copri before departure and after return. The composition of microbiota, before travel as well as at return, was not correlated with the acquisition of MRE. However, the clearance of MRE one month after return was linked to a specific pattern of bacterial species that was also found before and after return. In conclusion, we found specific OTUs associated to a higher risk of diarrhea during a stay in tropical regions and to a faster clearance of MRE after their acquisition.