Highly reliable GIGA-sized synthetic human therapeutic antibody library construction.

Background: Monoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library's potential for biomedical applications. Methods: The library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to ß-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays. Results: We have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation. Conclusion: The promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies.

Neutrophil Extracellular Traps Impair Intestinal Barrier Function during Experimental Colitis.

Aberrant neutrophil extracellular trap (NET) formation and the loss of barrier integrity in inflamed intestinal tissues have long been associated with inflammatory bowel disease (IBD). However, whether NETs alter intestinal epithelium permeability during colitis remains elusive. Here, we demonstrated that NETs promote the breakdown in intestinal barrier function for the pathogenesis of intestinal inflammation in mouse models of colitis. NETs were abundant in the colon of mice with colitis experimentally induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). Analysis of the intestinal barrier integrity revealed that NETs impaired gut permeability, enabling the initiation of luminal bacterial translocation and inflammation. Furthermore, NETs induced the apoptosis of epithelial cells and disrupted the integrity of tight junctions and adherens junctions. Intravenous administration of DNase I, an enzyme that dissolves the web-like DNA filaments of NETs, during colitis restored the mucosal barrier integrity which reduced the dissemination of luminal bacteria and attenuated intestinal inflammation in both DSS and TNBS models. We conclude that NETs serve a detrimental factor in the gut epithelial barrier function leading to the pathogenesis of mucosal inflammation during acute colitis.

α-Linolenic acid inhibits the migration of human triple-negative breast cancer cells by attenuating Twist1 expression and suppressing Twist1-mediated epithelial-mesenchymal transition.

α-Linolenic acid (ALA), an essential fatty acid, has anticancer activity in breast cancer, but the mechanism of its effects in triple-negative breast cancer (TNBC) remains unclear. We investigated the effect of ALA on Twist1, which is required to initiate epithelial-mesenchymal transition (EMT) and promotes tumor metastasis, and Twist1-mediated migration in MDA-MB231, MDA-MB468 and Hs578T cells. Twist1 protein was constitutively expressed in these TNBC cells, particularly MDA-MB-231 cells. Treatment with 100 µM ALA and Twist1 siRNA markedly decreased the Twist1 protein level and cell migration. Moreover, ALA transiently attenuated the nuclear accumulation of STAT3α as well as Twist1 mRNA expression. Treatment with ALA significantly attenuated the phosphorylation of JNK, ERK and Akt and decreased the phosphorylation of Twist1 at serine 68 in MDA-MB-231 cells. ALA accelerated Twist1 degradation in the presence of cycloheximide, whereas the ubiquitination and degradation of Twist1 by ALA was suppressed by MG-132. Pretreatment with ALA mimicked Twist1 siRNA, increased the protein expression of epithelial markers such as E-cadherin, and decreased the protein expression of mesenchymal markers including Twist1, Snail2, N-cadherin, vimentin, and fibronectin. Our findings suggest that ALA can be used not only to abolish EMT but also to suppress Twist1-mediated migration in TNBC cells.

Application of stepper in cardiopulmonary exercise test for patients with hemiplegia.

A method to perform exercise testing for patients with hemiplegia is unavailable though over half of them have cardio-pulmonary disorders. We aimed to assess the reliability and validity of using a stepper in cardiopulmonary exercise testing (CPET) in this population.14 stroke patients with hemiplegia who failed to ride the stationary bike were included. Exclusion criteria included manual muscle strength ≦1 in the lower extremity, and conventional contraindications of CPET. They underwent CPET twice by using a stepper to evaluate test-retest reliability and validity. Additionally, 10 healthy participants underwent CPET twice on the cycle ergometer and stepper respectively.In the test-retest, the ratio of two-time difference to mean was 5.0, 3, 11.3 and 12.0% on average for peak oxygen consumption, peak heart rate (HR), anaerobic threshold and minute ventilation - carbonic dioxide production slope respectively. Cronbach's alpha coefficient of peak oxygen consumption and anaerobic threshold were 0.992 and 0.919. In the stepper exercise testing of the hemiplegic participants, the ratio of peak HR to age-predicted maximal HR was 75% on average. Peak respiratory exchange ratio (mean ±â€Šstandard deviation = 1.17 ±â€Š0.08) was not different from that of healthy controls (1.21 ±â€Š0.09). Notably, VO2 trajectory in relation to work rate is nonlinear and different in the rest-retest.This is the first research to study CPET variables in detail using stepper in patients with hemiplegia. CPET variables associated with peak are valid and reliable; nonetheless, those with sub-maximum are not. The study provides a method to do exercise testing for the patients with hemiplegia and its notice in application.

A systematic review of dietary acids and habits on dental erosion in adolescents.

BACKGROUND: Dental erosion is the dissolution of dental hard tissues caused by acids of a non-bacterial origin. Dietary acids are considered the predominant and most controllable factor. AIM: To synthesise the literature on the effects of dietary acids and habits on dental erosion in the permanent dentition of 10- to 19-year-old adolescents. MATERIALS AND METHODS: An electronic literature search was undertaken in Cochrane, MEDLINE, CINAHL, Dentistry & Oral Sciences Source via EBSCOhost, and Embase with no restriction on the date of publication. RESULTS: The initial search identified 449 articles, and 338 remained after removal of duplicates. Seventy-seven articles remained after screening of titles and abstracts, and 52 were eligible for the full-text review. A considerable variety of beverages, food, and dietary habits were reported as risk factors for dental erosion. The most consistent findings implicated the erosive potential of carbonated beverages and the consumption of acidic drinks at bedtime. CONCLUSIONS: Although results were not consistent between cohort and cross-sectional studies, this review suggests certain dietary risk factors may contribute to dental erosion in adolescents. There is a need for more high-quality cohort studies to establish more conclusive evidence on the role of dietary acids and habits on dental erosion.

Microtubule-Mediated NLRP3 Inflammasome Activation Is Independent of Microtubule-Associated Innate Immune Factor GEF-H1 in Murine Macrophages.

Inflammasomes are intracellular multiple protein complexes that mount innate immune responses to tissue damage and invading pathogens. Their excessive activation is crucial in the development and pathogenesis of inflammatory disorders. Microtubules have been reported to provide the platform for mediating the assembly and activation of NLRP3 inflammasome. Recently, we have identified the microtubule-associated immune molecule guanine nucleotide exchange factor-H1 (GEF-H1) that is crucial in coupling microtubule dynamics to the initiation of microtubule-mediated immune responses. However, whether GEF-H1 also controls the activation of other immune receptors that require microtubules is still undefined. Here we employed GEF-H1-deficient mouse bone marrow-derived macrophages (BMDMs) to interrogate the impact of GEF-H1 on the activation of NLRP3 inflammasome. NLRP3 but not NLRC4 or AIM2 inflammasome-mediated IL-1ß production was dependent on dynamic microtubule network in wild-type (WT) BMDMs. However, GEF-H1 deficiency did not affect NLRP3-driven IL-1ß maturation and secretion in macrophages. Moreover, α-tubulin acetylation and mitochondria aggregations were comparable between WT and GEF-H1-deficient BMDMs in response to NLRP3 inducers. Further, GEF-H1 was not required for NLRP3-mediated immune defense against Salmonella typhimurium infection. Collectively, these findings suggest that the microtubule-associated immune modulator GEF-H1 is dispensable for microtubule-mediated NLRP3 activation and host defense in mouse macrophages.

Protein Glycation by Glyoxal Promotes Amyloid Formation by Islet Amyloid Polypeptide.

Protein glycation, also known as nonenzymatic glycosylation, is a spontaneous post-translational modification that would change the structure and stability of proteins or hormone peptides. Recent studies have indicated that glycation plays a role in type 2 diabetes (T2D) and neurodegenerative diseases. Over the last two decades, many types of advanced glycation end products (AGEs), formed through the reactions of an amino group of proteins with reducing sugars, have been identified and detected in vivo. However, the effect of glycation on protein aggregation has not been fully investigated. In this study, we aim to elucidate the impact of protein glycation on islet amyloid polypeptide (IAPP, also known as amylin) aggregation, which was strongly associated with T2D. We chemically synthesized glycated IAPP (AGE-IAPP) to mimic the consequence of this hormone peptide in a hyperglycemia (high blood sugar) environment. Our data revealed that AGE-IAPP formed amyloid faster than normal IAPP, and higher-molecular-weight AGE-IAPP oligomers were also observed in the early stage of aggregation. Circular dichroism spectra also indicated that AGE-IAPP exhibited faster conformational changes from random coil to its ß-sheet fibrillar states. Moreover, AGE-IAPP can induce normal IAPP to expedite its aggregation process, and its fibrils can also act as templates to promote IAPP aggregation. AGE-IAPP, like normal IAPP, is capable of interacting with synthetic membranes and also exhibits cytotoxicity. Our studies demonstrated that glycation modification of IAPP promotes the amyloidogenic properties of IAPP, and it may play a role in accumulating additional amyloid during T2D progression.

Non-invasive assessment determine the swallowing and respiration dysfunction in early Parkinson's disease.

INTRODUCTION: Dysphagia is common among patients with Parkinson's disease. Swallowing and its coordination with respiration is extremely important to achieve safety swallowing. Different tools have been used to assess this coordination, however the results have been inconsistent. We aimed to investigate this coordination in patients with Parkinson's disease using a non-invasive method. METHODS: Signals of submental muscle activity, thyroid cartilage excursion, and nasal airflow during swallowing were recorded simultaneously. Five different water boluses were swallowed three times, and the data were recorded and analyzed. RESULTS: Thirty-seven controls and 42 patients with early-stage Parkinson's disease were included. The rates of non-expiratory/expiratory pre- and post-swallowing respiratory phase patterns were higher in the patients than in the controls (P < 0.001). The rates of piecemeal deglutition when swallowing 10-ml and 20-ml water boluses and overall were also significantly higher in the patients (all P < 0.001). There were differences in oropharyngeal swallowing parameters between the patients and controls, including a pharyngeal phase delay with longer total excursion duration and excursion time in the patients swallowing small water boluses (1 ml, 3 ml and 5 ml), but no difference in the length of swallowing respiratory pause. CONCLUSION: Oropharyngeal swallowing and its coordination with respiration are affected in patients with early-stage Parkinson's disease, and safety compensation mechanisms were used more than efficiency during swallowing. The results of this study may serve as a baseline for further research into new treatment regimens and to improve the management of swallowing in patients with Parkinson's disease.

Molecular recircumscription of Broussonetia (Moraceae) and the identity and taxonomic status of B. kaempferi var. australis.

BACKGROUND: Despite being a relatively small genus, the taxonomy of the paper mulberry genus Broussonetia remains problematic. Much of the controversy is related to the identity and taxonomic status of Broussonetia kaempferi var. australis, a name treated as a synonym in the floras of Taiwan and yet accepted in the floras of China. At the generic level, the monophyly of Corner (Gard Bull Singap 19:187-252, 1962)'s concept of Broussonetia has not been tested. In recent studies of Broussonetia of Japan, lectotypes of the genus were designated and three species (B. kaempferi, Broussonetia monoica, and Broussonetia papyrifera) and a hybrid (B. ×kazinoki) were recognized. Based on the revision and molecular phylogenetic analyses, this article aims to clarify these issues. RESULTS: Herbarium studies, field work, and molecular phylogenetic analyses indicate that all Taiwanese materials identifiable to B. kaempferi var. australis are conspecific with B. monoica of Japan and China. Molecular phylogenetic analyses showed that Broussonetia sensu Corner (Gard Bull Singap 19:187-252, 1962) contains two clades corresponding to sect. Broussonetia and sect. Allaeanthus, with Malaisia scandens sister to sect. Broussonetia. CONCLUSIONS: Based on our analyses, B. kaempferi var. australis is treated as a synonym of B. monoica and that B. kaempferi is not distributed in Taiwan. To correct the non-monophyly of Broussonetia sensu Corner (Gard Bull Singap 19:187-252, 1962), Broussonetia is recircumscribed to contain only sect. Broussonetia and the generic status of Allaeanthus is reinstated.