Twenty years of "Lipid World": a fertile partnership with David Deamer

"The Lipid World" was published in 2001, stemming from a highly effective collaboration with David Deamer during a sabbatical year 20 years ago at the Weizmann Institute of Science in Israel. The present review paper highlights the benefits of this scientific interaction and assesses the impact of the lipid world paper on the present understanding of the possible roles of amphiphiles and their assemblies in the origin of life. The lipid world is defined as a putative stage in the progression towards life's origin, during which diverse amphiphiles or other spontaneously aggregating small molecules could have concurrently played multiple key roles, including compartment formation, the appearance of mutually catalytic networks, molecular information processing, and the rise of collective self-reproduction and compositional inheritance. This review brings back into a broader perspective some key points originally made in the lipid world paper, stressing the distinction between the widely accepted role of lipids in forming compartments and their expanded capacities as delineated above. In the light of recent advancements, we discussed the topical relevance of the lipid worldview as an alternative to broadly accepted scenarios, and the need for further experimental and computer-based validation of the feasibility and implications of the individual attributes of this point of view. Finally, we point to possible avenues for exploring transition paths from small molecule-based noncovalent structures to more complex biopolymer-containing proto-cellular systems.711473 - Minerva Foundation; 80NSSC17K0295, 80NSSC17K0296, 1724150 - National Science FoundationPublished versio

Genomic Dissection and Expression Profiling Revealed Functional Divergence in Triticum aestivum Leucine Rich Repeat Receptor Like Kinases (TaLRRKs)

The leucine rich repeat receptor like kinases (LRRK) constitute the largest subfamily of receptor like kinases (RLK), which play critical roles in plant development and stress responses. Herein, we identified 531 TaLRRK genes in Triticum aestivum (bread wheat), which were distributed throughout the A, B, and D sub-genomes and chromosomes. These were clustered into 233 homologous groups, which were mostly located on either homeologous chromosomes from various sub-genomes or in proximity on the same chromosome. A total of 255 paralogous genes were predicted which depicted the role of duplication events in expansion of this gene family. Majority of TaLRRKs consisted of trans-membrane region and localized on plasma-membrane. The TaLRRKs were further categorized into eight phylogenetic groups with numerous subgroups on the basis of sequence homology. The gene and protein structure in terms of exon/intron ratio, domains and motifs organization were found to be variably conserved across the different phylogenetic groups/subgroups, which indicated a potential divergence and neofunctionalization during evolution. High-throughput transcriptome data and quantitative real time PCR analyses in various developmental stages, and biotic and abiotic (heat, drought and salt) stresses provided insight into modus operandi of TaLRRKs during these conditions. Distinct expression of majority of stress responsive TaLRRKs homologous genes suggested their specified role in a particular condition. These results provided a comprehensive analysis of various characteristic features including functional divergence, which may provide the way for future functional characterization of this important gene family in bread wheat

Cartilage tissue engineering: uses of injection molding and computer aided design for the fabrication of complex geometries with high dimensional tolerances: a dissertation

Cartilage Tissue Engineering. Joint pain and functional impairment due to cartilage damage from osteoarthritis and other means is a major source of disability for adults the world over. Cartilage is an avascular tissue with a very limited capacity for self repair. Current medical and surgical approaches to cartilage repair also have limited efficacy, and in all cases fail to completely restore a normal, healthy cartilage phenotype. Tissue engineering is a relatively new approach to cartilage repair that seeks to fabricate a replacement tissue, indistinguishable from healthy, native tissue. The basic idea of the tissue engineering approach is to seed tissue synthesizing cells into a shapeable, biocompatible/bioabsorbable scaffold that serves as a temporary extracellular matrix with a localized source of bioactive molecules to direct the development of new tissue. The challenge of tissue engineering is to identify cells, scaffolds, and growth conditions that will be optimal for tissue regeneration. The goal of the current studies was to evaluate one aspect of all three of the major components of cartilage tissue engineering: cell source, scaffolding material and preparation, and controlled growth factor delivery. We evaluated the chondrogenic potential of human nasal chondrocytes grown in calcium alginate in an in vivo culture system, the potential of computer-aided design and injection molding with calcium alginate to reliably reproduce complex geometries with high dimensional tolerances, and the potential for the controlled release of TGF-β1 from calcium alginate modified by the covalent addition of a recently discovered TGF-β binding peptide. We found that adult human nasal chondrocytes show significant chondrogenic potential when grown within an alginate scaffold. We also found that alginate is readily amenable to an injection molding process that utilizes precision made molds from computer-aided design and solid free form fabrication, allowing for the fabrication of tissue engineered constructs with very precise shape fidelity. Additionally, we found that calcium alginate could be reliably modified by the covalent addition of peptides, and that the addition of a newly discovered TGF-β binding peptide delayed the release of pre-loaded TGF-β1. Together these results show some of the encouraging prospects for cartilage tissue engineering. `Menière’s Syndrome.Menière’s syndrome is an inner ear disorder characterized by idiopathic endolymphatic hydrops with associated periodic tinnitus, vertigo, and progressive sensorineural hearing loss. It affects approximately 0.2% of the population, for whom it can be quite devastating. In addition to progressive hearing loss people with Menière’s syndrome are prone to sudden attacks of vertigo and tinnitus that are severe enough that they can lead to falls and potentially serious injury. People subject to frequent attacks are unable to drive, with obvious consequences on standard of living. In the current studies we evaluated the standard animal model of Menière’s syndrome by comparing cochlear turn specific hearing thresholds and the degree of hydrops in that turn. A positive correlation between these had previously been established in the study of human temporal bones from people with Menière’s syndrome, but had not been reported in the animal model. We also evaluated the potential of aminoguanidine, a relatively specific inhibitor of the inducible isoform of nitric oxide synthase, as a neuroprotective therapeutic agent for preservation of hearing in animals with surgically induced endolymphatic hydrops. We found, for the first time, a partial correlation between cochlear turn specific hydrops and hearing thresholds in the most commonly used animal model of Menière’s syndrome, helping to validate the utility of this animal model for future studies. We also found that aminoguanidine did indeed partially preserve hearing in animals with surgically induced Menière’s syndrome. This encouraging result appears to be the first report of a medical intervention protective against hearing loss in an animal model of Menière’s syndrome, and may help us to understand the etiology pathology seen in Menière’s syndrome

단분자 자기 집게 기술을 이용한 당 수송 막단백질의 접힘 및 진화과정 연구

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 생명과학부, 2023. 2. 윤태영.Despite advances in resolving structures of multi-pass membrane proteins, little is known about the native folding pathways of these complex structures. Using single-molecule magnetic tweezers, I found a folding pathway of purified human glucose transporter 3 (GLUT3) reconstituted within synthetic lipid bilayers. The N-terminal major facilitator superfamily (MFS) fold strictly forms first, serving as structural templates for its C-terminal counterpart. Based on structure, polar residues comprising the conduit for glucose molecules present major folding challenges. The ER membrane protein complex facilitates insertion of these hydrophilic transmembrane helices, thrusting GLUT3s microstate sampling toward folded structures. Final assembly between the N- and C-terminal MFS folds depends on specific lipids that ease desolvation of lipid shells surrounding the domain interfaces. Sequence analysis suggests that this asymmetric folding propensity across the N- and C-terminal MFS folds prevails for metazoan sugar porters, revealing evolutionary conflicts between foldability and functionality faced by many multi-pass membrane proteins.세포 내에서 일어나는 여러 현상은 막으로 구분되어 있으며, 이러한 막에 존재하는 막 단백질들은 물질을 수송하거나 정보를 전달하는 등 매우 중요한 역할을 담당한다. 이러한 막 단백질이 기능을 하기 위해서는 올바른 접힘 과정을 통해 구조가 형성되어야 한다. 그런데 완성된 구조에 대한 연구는 많이 진행되고 있지만 이러한 구조의 형성 과정에 대한 연구는 부족하다. 막 단백질의 구조 형성과정은 단백질이 인지질 이중막에서 전사되는 과정과 매우 밀접하게 관련이 있다. 이러한 세포내 환경을 모사하고 막 단백질의 풀림, 접힘 과정을 살펴보기 위해 자기 집게 기술 (single-molecule magnetic tweezers)을 이용하였다. 이 기술을 통해 막 단백질에 pN 단위의 미세한 힘을 가할 수 있다. 목표로 한 막 단백질은 복잡한 당 수송 단백질인 GLUT3로 해당 막 단백질의 접힘 과정을 최초로 규명하였다. 특히 소포체막 단백질 복합체(EMC)와 특이구조를 지닌 지질분자를 이용하여 생리학적인 환경에서 막 단백질의 구조 형성 과정을 완전히 밝혀냈다. 이를 통해 막 단백질이 다른 단백질, 그리고 인지질과 같은 주변 환경의 도움을 통해 기능할 수 있는 구조를 형성함을 알아냈다. 이러한 결과를 확장하고자 생물정보학을 이용하여 다양한 당 수송 단백질의 서열을 분석하였다. 당 수송 단백질은 6개의 transmembrane helix로 이루어진 domain 2개가 존재하는데 이러한 사실과 생물정보학 분석을 종합한 결과, 각각의 domain이 단백질의 구조형성 능력과 새로운 수송 기능을 담당하며 균형을 맞추어 진화해 왔음을 발견하였다.Abstract i Chapter 1. Introduction １ 1.1. Folding of helical membrane proteins １ 1.2. Biogenesis of membrane proteins ３ 1.3. Major facilitator superfamily (MFS) ４ 1.4. Single-molecule magnetic tweezers ５ Chapter 2. Methodology ７ 2.1. Sample Preparation ７ 2.1.1. Expression and purification of the human GLUT3 ７ 2.1.2. Expression and purification of the human ER membrane protein complex １０ 2.1.3. Preparation of DNA handles １２ 2.1.4. Preparation of bicelle １３ 2.2. Experimental Methods １４ 2.2.1. Dynamic light scattering (DLS) measurement １４ 2.2.2. single-molecule magnetic tweezers experiments １４ 2.2.3. Molecular dynamics simulations １６ 2.2.4. Sequence alignment and determination of helix insertion energy １８ 2.3. Data Analysis ２０ 2.3.1. Force-extension curves (FEC) analysis ２０ 2.3.2. Hidden Markov Model (HMM) analysis ２２ 2.3.3. Deconvoluted extension probability analysis ２２ Chapter 3. Results ２５ 3.1. Single-molecule magnetic tweezers monitoring GLUT3 folding ２５ 3.2. Mapping the folding order of single GLUT3 domains ２９ 3.3. Dissecting folding steps of the MFS folds ３２ 3.4. EMC facilitates insertion of TMHs of GLUT3 ３６ 3.5. PE lipids boost domain-domain assembly of GLUT3 ４０ 3.6. Asymmetric TMH distributions of metazoan sugar transporters ４４ Chapter 4. Conclusion ４８ Supplementary figure ５１ Bibliography ６３ Abstract in Korean (초록) ７０ 감사의 글 ７１박

Forward and inverse metabolic engineering strategies for improving polyhydroxybyrate production

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2008.Includes bibliographical references (p. 165-174).Forward metabolic engineering (FME) is a rational approach to cellular engineering, relying on an understanding of the entire metabolic network to direct perturbations for phenotype improvement. Conversely, inverse metabolic engineering (IME) uses a global, combinatorial approach to identify genetic loci that are important for a given phenotype. These two approaches complement each other in a strain improvement program. FME and IME approaches were applied to poly-3-hydroxybutyrate (PHB)production in Synechocystis PCC6803 [IME] and recombinant E. coli [FME] in this thesis.IME was appropriate for Synechocystis, where metabolic regulation of the native PHB pathway was not well understood. A high throughput screening method was established by developing a staining protocol that quantitatively related nile red fluorescence to PHB content, while maintaining cell viability for both organisms. This was combined with fluorescence activated cell sorting (FACS) to screen for high PHB mutants. A Synechocystis insertion mutagenesis library was screened to identify gene disruptions that increased PHB. Two gene disruptions in proline biosynthesis and an unknown function were identified and characterized.An analogous IME study in E. coli did not find increased PHB mutants, but suggested an FME approach on the PHB pathway. Systematic overexpression of the pathway revealed phaB, acetoacetyl-CoA reductase, limited PHB flux. Beyond this, whole operon overexpression led to even higher PHB fluxes.In a nitrogen-limited chemostat, PHB flux did not change with dilution rate. Unlike prior pleiotropic perturbations, these systematic experiments could clearly conclude that the flux control is in the PHB pathway. At high PHB flux, growth rate was extremely hindered and was accompanied by PHB plasmid genetic instability and rapid PHB productivity loss.(cont.) Tandem gene duplication (TGD) was developed to slow productivity loss caused by "allele segregation," a fast process that propagates a DNA mutation to all copies of a plasmid. By placing the many copies in tandem, rather than on individual plasmids, allele segregation could be avoided, increasing stability significantly.These methods and results should support PHB engineering in higher photosynthetic organisms and better E. coli PHB production in batch or continuous culture.TGD is a broadly applicable technique for high level recombinant expression.by Keith E. J. Tyo.Ph.D

Transforming semi-structured life science diagrams into meaningful domain ontologies with DiDOn

AbstractBio-ontology development is a resource-consuming task despite the many open source ontologies available for reuse. Various strategies and tools for bottom-up ontology development have been proposed from a computing angle, yet the most obvious one from a domain expert perspective is unexplored: the abundant diagrams in the sciences. To speed up and simplify bio-ontology development, we propose a detailed, micro-level, procedure, DiDOn, to formalise such semi-structured biological diagrams availing also of a foundational ontology for more precise and interoperable subject domain semantics. The approach is illustrated using Pathway Studio as case study

Review on bibliography related to antimicrobials

In this report, a bibliographic research has been done in the field of antimicrobials.In this report, a bibliographic research has been done in the field of antimicrobials. Not all antimicrobials have been included, but those that are being subject of matter in the group GBMI in Terrassa, and others of interest. It includes chitosan and other biopolymers. The effect of nanoparticles is of great interest, and in this sense, the effect of Ag nanoparticles and antibiotic nanoparticles (nanobiotics) has been revised. The report focuses on new publications and the antimicrobial effect of peptides has been considered. In particular, the influence of antimicrobials on membranes has deserved much attention and its study using the Langmuir technique, which is of great utility on biomimetic studies. The building up of antimicrobials systems with new techniques (bottom-up approach), as the Layer-by-Layer technique, can also be found in between the bibliography. It has also been considered the antibiofilm effect, and the new ideas on quorem sensing and quorum quenching.Preprin

New Frontiers in Synthetic Biology for Spaceflight

Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory