Deep Sequencing Analysis of Phage Libraries using Illumina Platform

This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 107 reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5′-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2 × 107 reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6 bp), one complimentary region (12 bp) and a variable region (36 bp). We applied this sequencing to a model library of 106 unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve diversity of phage clones and identify ligands previously lost in phage display screens.Chemistry and Chemical Biolog

Rho-kinase Regulates Energy Balance by Targeting Hypothalamic Leptin Receptor Signaling

Leptin regulates energy balance. However, knowledge of the critical intracellular transducers of leptin signaling remains incomplete. Here we report that Rho-kinase 1 (ROCK1) regulates leptin action on body weight homeostasis by activating JAK2, an initial trigger of leptin receptor signaling. Leptin promotes the physical interaction of JAK2 and ROCK1, thereby increasing phosphorylation of JAK2 and downstream activation of Stat3 and FOXO1. Mice lacking ROCK1 in either POMC or AgRP neurons, mediators of leptin action, display obesity and impaired leptin sensitivity. In addition, deletion of ROCK1 in the arcuate nucleus markedly enhances food intake, resulting in severe obesity. Of note, ROCK1 is a specific mediator of leptin, but not insulin, regulation of POMC neuronal activity. Our data identify ROCK1 as a key regulator of leptin action on energy homeostasis

Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53

The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses

PDMS-ENCAPSULATED CRACK SENSOR INTEGRATED WITH SILICON RUBBER CANTILEVER FOR USE IN CELL CULTURE MEDIA

In this study, we propose a novel cantilever structure integrated with a high-sensitive and-reliable crack sensor that monitors the contractile behavior of cardiomyocytes in media. Si rubber with excellent performance in cell adhesiveness was used as the cantilever material. The proposed crack sensor formed on the Si rubber cantilever is chemically bonded with the PDMS thin layer to produce a sandwich structure. The protection layer greatly improves the reliability and stability of the crack sensor in the electrolyte solution. The high-sensitive crack sensor stably measures the contractility of cardiomyocytes without changing a gauge factor for up to 26 days.N

27503265

BACKGROUND CONTEXT:Generalized joint laxity (GJL) can have a negative impact on lumbar spine pathology, including low back pain, disc degeneration, and disc herniation, but the relationship between GJL and cervical spine conditions remains unknown.PURPOSE:To investigate the relationship between GJL and cervical spine conditions, including the prevalence of posterior neck pain (PNP), cervical disc herniation (CDH), and cervical disc degeneration (CDD), in a young, active population.STUDY DESIGN:Retrospective 1:2 matched cohort (case-control) study from prospectively collected data PATIENT SAMPLE: Of a total of 1853 individuals reviewed, 73 individuals with GJL (study group, gruop A) and 146 without GJL (control group, Group B) were included in the study according to a 1:2 case-control matched design for age, sex, and body mass index.OUTCOME MEASURE:The primary outcome measure was the prevalence and intensity of PNP at enrollment based on a visual analogue scale score for pain. The secondary outcome measures were (1) clinical outcomes as measured with the neck disability index (NDI) and 12-item short form health survey (SF-12) at enrollment, and (2) radiological outcomes of CDH and CDD at enrollment.METHODS:We compared baseline data between groups. Descriptive statistical analyses were performed to compare the 2 groups in terms of the outcome measures.RESULTS:The prevalence and intensity of PNP were significantly greater in group A (patients with GJL) than in group B (patients without GJL) (prevalence: p=.02; intensity: p=.001). Clinical outcomes as measured with NDI and SF-12 did not differ significantly between groups. For radiologic outcomes, the prevalence of CDD was significantly greater in group A than in group B (p=.04), whereas the prevalence of CDH did not differ significantly between groups (p=.91).CONCLUSIONS:The current study revealed that GJL was closely related to the prevalence and intensity of PNP, suggesting that GJL may be a causative factor for PNP. In addition, GJL may contribute to the occurrence of CDD, but not CDH. Spine surgeons should screen for GJL in patientswith PNP and inform patients of its potential negative impact on disc degeneration of the cervical spine.OAIID:RECH_ACHV_DSTSH_NO:T201700367RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A079510CITE_RATE:2.66FILENAME:TSJ-2016_Lee_The impact of generalized joint laxity (GJL) on the posterior neck.pdfDEPT_NM:의학과EMAIL:[email protected]_YN:YFILEURL:https://srnd.snu.ac.kr/eXrepEIR/fws/file/db707bc2-2eae-4527-841f-2f0fc699192c/linkCONFIRM:

Isolation of Bacteriophages Which Can Infect Pectobacteirum carotovorum subsp. carotovorum

International audienc

Growth of Hexagonal Germanium Microcrystal Using Al-Based Nanostructures

우리는 알루미늄 기반 나노구조 (Al-based nanostructure)를 통해 대기압에서 육각형 (hexagonal) 게르마늄(germanium, Ge) 마이크로 결정 성장에 대한 새로운 접근 방식을 제안한다. 특히 Ge은 자연 입방체 상에서 간접적인 밴드갭 에너지를 갖지만, 육각형 상 (hexagonal phase)은 준 직접 밴드갭 (quasi-direct bandgap) 에너지를 가지므로 새로운 고속 전자 및 광자 장치에 대한 새로운 기회를 열 수 있다. 우리는 자연에서 거미줄에 물방울이 형성되는 것과 같이 나노 구조에 의한 Ge 구슬의 발생을 보고한다. 이를 통하여 육각형 Ge 마이크로 결정을 구현하였다. 본 논문에서는 알루미늄 기반 나노 구조의 형성과정과 특성 및 역할을 보고하고, 이 방법에 의해 성장된 육각형 Ge 마이크로 결정은 주사전자현미경 (FE-SEM), EDS 및 Raman 분광법을 사용하여 확인되었다. 따라서 이 방법은 자연 현상과 유사한 현상으로 설명할 수 있는 반도체 성장 분야의 적절한 예가 될 것이다. We propose a novel approach for growing hexagonal germanium (Ge) at atmospheric pressure using an Al-based nanostructure. In particular, Ge features an indirect bandgap in the natural cubic phase but a quasi-direct bandgap in the hexagonal phase, which opens up new opportunities for new high-speed electronic and photonic devices. We investigated the growth of hexagonal Ge microcrystals using nanowires. The formation process, characteristics, and role of Al-based nanostructure were investigated, and hexagonal Ge grown via this method was confirmed by field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Raman spectroscopy. Therefore, this technique, which mimics natural phenomena, will be useful in the field of semiconductor growth. © 2023 The Korean Physical Society. All rights reserved.TRU