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23.16: Complement System - Biology

23.16: Complement System - Biology


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An array of approximately 20 types of soluble proteins, called a complement system, functions to destroy extracellular pathogens. After the first few complement proteins bind, a cascade of sequential binding events follows in which the pathogen rapidly becomes coated in complement proteins.

Complement proteins perform several functions. The proteins serve as a marker to indicate the presence of a pathogen to phagocytic cells, such as macrophages and B cells, and enhance engulfment; this process is called opsonization. Opsonization refers to an immune process where particles such as bacteria are targeted for destruction by an immune cell known as a phagocyte. Certain complement proteins can combine to form attack complexes that open pores in microbial cell membranes. These structures destroy pathogens by causing their contents to leak, as illustrated in Figure 1.

Figure 1. Click for a larger image. The classic pathway for the complement cascade involves the attachment of several initial complement proteins to an antibody-bound pathogen followed by rapid activation and binding of many more complement proteins and the creation of destructive pores in the microbial cell envelope and cell wall. The alternate pathway does not involve antibody activation. Rather, C3 convertase spontaneously breaks down C3. Endogenous regulatory proteins prevent the complement complex from binding to host cells. Pathogens lacking these regulatory proteins are lysed. (credit: modification of work by NIH)


Embryo ecology: Developmental synchrony and asynchrony in the embryonic development of wild annual fish populations

Matej Polačik, Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic.

Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), Funding acquisition (equal), ​Investigation (equal), Methodology (equal), Project administration (equal), Supervision (equal), Visualization (equal), Writing - original draft (equal), Writing - review & editing (equal)

Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic

Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), ​Investigation (equal), Methodology (equal), Visualization (equal), Writing - original draft (equal), Writing - review & editing (equal)

Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic

Contribution: ​Investigation (equal), Methodology (equal), Supervision (equal), Writing - original draft (equal)

Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic

Department of Zoology, Charles University, Prague, Czech Republic

Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), ​Investigation (equal), Methodology (equal), Visualization (equal), Writing - original draft (equal)

Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic

Contribution: Conceptualization (equal), ​Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal)

Center for Life in Extreme Environments, Portland State University, Portland, OR, USA

Contribution: Conceptualization (equal), ​Investigation (equal), Methodology (equal), Resources (equal), Validation (equal), Visualization (equal), Writing - original draft (equal), Writing - review & editing (equal)

Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic

Matej Polačik, Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic.

Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), Funding acquisition (equal), ​Investigation (equal), Methodology (equal), Project administration (equal), Supervision (equal), Visualization (equal), Writing - original draft (equal), Writing - review & editing (equal)

Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic

Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), ​Investigation (equal), Methodology (equal), Visualization (equal), Writing - original draft (equal), Writing - review & editing (equal)

Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic

Contribution: ​Investigation (equal), Methodology (equal), Supervision (equal), Writing - original draft (equal)

Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic

Department of Zoology, Charles University, Prague, Czech Republic

Contribution: Conceptualization (equal), Data curation (equal), Formal analysis (equal), ​Investigation (equal), Methodology (equal), Visualization (equal), Writing - original draft (equal)

Institute of Vertebrate Biology, The Czech Academy of Sciences, Brno, Czech Republic

Contribution: Conceptualization (equal), ​Investigation (equal), Methodology (equal), Writing - original draft (equal), Writing - review & editing (equal)

Center for Life in Extreme Environments, Portland State University, Portland, OR, USA

Contribution: Conceptualization (equal), ​Investigation (equal), Methodology (equal), Resources (equal), Validation (equal), Visualization (equal), Writing - original draft (equal), Writing - review & editing (equal)


Medical biochemistry (4th ed.): Bhagavan, N. V.

Bhagavan, N. V. Harcourt/Academic Press, 2002, 1016 pp., ISBN 0-12-095440-0, $79.95.

The first edition of Bhagavan's book appeared in 1974, and this most recent version is updated extensively. In addition to Professor Bhagavan, himself, there are seven major contributors plus a dozen or so reviewers. The presentation of topics is logical and traditional. Thus Chapter 1 begins with a discussion of the properties of water and acids, bases, and buffers. There is no gentle introduction we are thrown straight into it. In a way this characterizes the whole approach taken in the book it gives the information and the medical connections for the busy and mature medical student, with few frills or flowery apologia for having to understand this stuff.

The text is integrated moderately, but there is no doubt that it is biochemistry. Many students would find this approach very satisfactory, I think. They want to get on and learn the important material and see its relevance. The division of topics between the chapters is also designed for medics. Thus the vitamins are dealt with in a separate chapter rather than being integrated into metabolism etc., and the hormones are divided into separate chapters on steroids, hypothalamus, and pituitary, adrenals, thyroid, and reproductive system, rather than according to their biochemical mode of action (e.g. G-protein interactions, intracellular zinc finger receptors).

The diagrams, too, are on the simple side for the most part. No artist has been let loose with a four-color palette this is all functional. In fact the book is printed mostly in back and white with the addition of some red used sparingly in some of the diagrams. However, there is a set of color pictures on glossy paper grouped together in a separate section (presumably as an economy measure) relating to Chapters 35 and 36, on molecular immunology and hemostasis, which are written by one of the contributing authors. These figures also appear in the appropriate place in the text as (rather ghostly) black and white images, but then you can turn to these extra pages for the color versions. These are mostly schemes and molecular structures. In fact I am not sure to what extent medics really need to know the three-dimensional structures of proteins (there is no CD-ROM with this book). For example under thyroid we have some curious and rather old molecular structures of T4 (and this chapter is perhaps less up to date than the rest), and one might ask why we need three different structures for tacrolimus in the immunology chapter. I would have thought that micrographs and electron micrographs would, in general, have been more useful, and there are relatively few of these, and some are poor to the extent of being useless (e.g. the polysome in Figure 25–15). Some of the pictures borrowed from other publications have come out rather poorly, too they could have benefited from redrawing. Examples of these poor figures are the picture of liver structure on p.200 (surely very important for medics, but perhaps they get it in studies of anatomy) and Figures 23–16, 15–19, and 28–11, to name but a few.

The sequence of topics is as follows: amino acids, proteins, thermodynamics and kinetics, enzymes, carbohydrates, (digestion and absorption), glycolysis and the TCA cycle, glycogen, protein and amino acid metabolism, lipids, cholesterol, muscle contraction, metabolic homeostasis, DNA, etc. (now half way into the book), regulation of gene expression, nucleotide metabolism, hemoglobin, metabolism of iron and heme, endocrinology (as mentioned above), molecular immunology, hemostasis, mineral and vitamin metabolism, and finally water, electrolytes, and acid-base balance. At the end there are a number of appendices including ones on heights and weights and recommended daily allowances (United States, of course), enzymes of clinical importance, electrophoresis of serum proteins, hemoglobins, abbreviations, and clinical laboratory parameters. Each chapter has well chosen and mostly up to date, but quite short, lists of supplementary reading. Thus as early as p. 33 we encounter amyotrophic lateral sclerosis, nitric oxide, and septic shock. I would say that students would find it easy to find the things they want, and many students do indeed prefer the traditional approach. In most if not all of the topics covered, we very rapidly get to the medical relevance, and the supplementary reading gives rapid access to relevant articles. But the author/editor obviously has some problems with what to leave out. Under proteins there is some very good up to date material about cystic fibrosis, Creutzfeldt-Jakob disease, and Alzheimer's disease, etc., but then the basic stuff on protein structure still tells us about Sanger's reagent (1-fluoro-2,4-dinitrobenzene), which has not been used for 25 years.

Here are some comments on the way some items are dealt with. The chapter on oxidative phosphorylation is rather diffident about whether everyone now accepts the Mitchell hypothesis. There is good and up to date coverage of mitochondrial diseases but too little, in my opinion, about cytochrome P450 enzymes and detoxification. There is quite a lot of information on P450 enzymes hidden in the steroids chapters, which may again reflect the lack of integration between the various writers. Contrary to what is stated, the defect in Marfan's syndrome is known (it is in fibrillin). As usual with medical biochemistry texts there is probably too much on glycogen storage diseases, which the medical students will never encounter, but the section on G6PDH deficiency is good as are the sections on hemoglobin and hemoglobinopathies. Similarly the sections on diabetes are good and suitably up to date, especially on Type II and obesity. The coverage of the Human Genome project is slightly curious. The number of genes is quoted as 50–100,000, and the impression is given that the whole of HUGO was supported by “federal funds,” which might rile the chaps in Cambridge (England) and elsewhere who made a significant contribution. There seems to be no mention of websites. These are of course ephemeral, but nevertheless there are many good ones out there. For example, when it is said that there are lots of cytokines, students might be pointed in the direction of www.copingwithcytokines.de, to give but one example.

On the whole the text has an integrated style despite chapters being written by several different authors, but there are some exceptions. Thus the chapters on molecular immunology and hemostasis also have some curious medical vignettes or case problems, which are interesting, but the style does not fit with the rest of the book. These chapters are quite well written, although one appreciates that it represents a considerable challenge to deal with these medically important areas in a relatively small space. In some chapters, including these, there is a tendency to give all the information in (usually large) tables (for example all the components of the immune system and all of the components of the complement system). This is OK, and indeed many students may find this a useful way to have information collected together for memorizing, but it does get a little heavy sometimes and is not always supported fully by textual comments.

Overall I felt that this was a text that would be attractive to medical students. It contains a lot of basic biochemistry, but in most cases this is made relevant by appropriate reference to disease and diagnosis. It is not completely integrated either in terms of content or presentation, but this probably matters less to the students, given that they will be able to find the things they want easily (for reference or for reviewing for an exam), with most medically relevant biochemistry being covered.


Results

Trait analyses of litter mixtures

High scores of CWM1 (first PCA axis, Fig. 1a) were mainly determined by low N and Mg concentrations, but high lignin and polyphenol concentrations, C : N and lignin : N ratios. The two litter mixtures Alnus + Fraxinus (on the N and Mg-rich end) and Pistacia + Quercus (on the lignin and polyphenol-rich end) were the two extremes along CWM1. CWM2 was related to high P and Ca concentrations (with the highest scores for the Fraxinus + Pistacia mixture) as opposed to high cellulose concentrations (with the lowest scores for the Alnus + Quercus mixture). Rao1 (first PCA axis, Fig. 1b) separated litter mixtures according to increasing dissimilarity of N concentrations, as well as lignin : N and C : N ratios, among species within the mixture (e.g. Alnus + Pistacia). Rao2 was largely determined by increasing dissimilarity in lignin concentrations towards high scores (e.g. Alnus + Fraxinus + Quercus) and increasing dissimilarity in polyphenol concentrations towards low scores (e.g. Fraxinus + Pistacia).

Site-specific differences in environmental conditions and decomposer communities

Variation in environmental conditions (soil temperature and relative humidity, and soil parameters) between sites (regional scale) was larger than within-site variation (local scale), as blocks within sites generally clustered together (Fig. 2). The first PCA axis (Env1) was mainly determined by increasing soil temperature, Olsen P, and pH, but decreasing sand content. The second PCA axis (Env2) was defined by increasing soil C : N ratio but decreasing NH4 + availability. Total nematode abundance differed significantly among sites, ranging between 65·9 and 3·1 nematodes g −1 soil, but the functional diversity of the nematode communities was similar (Fig. S3). While the active microbial biomass did not differ among sites (overall mean of 5·5 μg CO2-C g −1 soil h −1 ), microbial functional diversity differed considerably (Fig. S4). The effect size of macrofauna presence on litter mass loss significantly differed between sites but was similar between the two litter mixtures evaluated (Fig. S5).

Litter C loss and N immobilization

When analysing our data as a factorial design, we identified litter mixture identity as the major factor influencing litter C and N loss (F9,180 = 89·68 and 217·03, respectively, P < 0·001 in both cases, Fig. 3). Although a significant litter mixture identity × site interaction was also found (C loss: F81,180 = 2·31, P < 0·001 N loss: F81,180 = 1·74, P = 0·001), Alnus + Fraxinus and Pistacia + Quercus depicted the highest and lowest losses of these two major elements, respectively, across all sites (Figs S6 and S7). Litter N loss was strongly related to litter mass remaining, as a polynomial regression with a quadratic fitting explained 87% of the variability in litter N loss across litter mixtures and sites (Fig. 4). This relationship showed that during the early stages of the decomposition process (up to c. 40% of initial mass loss), N was immobilized, that is, there was a net increase in the amount of litter N and thus negative litter N loss. Beyond this threshold, net N release started in the later stages of the decomposition process. The patterns of N immobilization and release also depended on the initial N concentration of the litter mixtures, as N immobilization mainly occurred in the mixtures with lower initial N concentration (Pistacia + Quercus and Fraxinus + Pistacia + Quercus).

Regional drivers of litter C and N loss during decomposition

Litter trait diversity had the largest influence on litter C and N loss in the structural equation models (Fig. 5), with both aspects of CWM traits and trait dissimilarity (Rao's Q) playing an important role. In the litter C model, the negative effect of CWM1 (r = −0·61, Fig. 5a) indicated higher litter C loss with higher litter N and Mg concentrations, but lower C : N and lignin : N ratios. Rao1 and Rao2 had a much smaller, although significant effect on litter C loss. The litter N model revealed a similar, even stronger, relationship with CWM1 (r = −0·70) compared to litter C loss (Fig. 5b). Remarkably, litter N loss was more affected by trait dissimilarity (Rao1 and Rao2) than C loss. In particular, Rao2 had a marked positive effect on litter N loss (r = 0·48), indicating that N loss increased with increasing within-mixture dissimilarity in lignin, N, lignin : N and C : N ratios, but decreasing dissimilarity in polyphenol concentrations. The role of CWM2 was very week in both models.

The site-specific environmental conditions accounting for variation in soil microclimate and physicochemistry (Env1 and Env2) had overall much less impact on C and N loss compared to the litter diversity mechanism (Fig. 5). In contrast, Env1 and Env2 both had a clear and strong impact on nematodes (abundance and functional diversity) and microbes (biomass and functional diversity). However, this influence did not translate into a similarly strong effect of these two groups of soil organisms on litter C and N loss. Consequently, the indirect effects of environmental conditions mediated by soil decomposers were of similar magnitude compared to the direct effects (Fig. S8). Interestingly, however, the effect of soil microbes on litter C loss was positive while it was negative for litter N loss. While nematodes had a strong direct effect on soil microbes, their contribution to litter decomposition was small, as indicated by their low indirect effects on C and N loss (Fig. S8). The macrofauna effect size on litter mass loss (lnRR fauna) was the most important driver of litter C and N loss after CWM and Rao's Q, with macrofauna promoting higher losses of litter C and N (r = 0·32 and 0·34).


Science in medicine: when, how, and what

February 27, 2014: This chapter has been re-evaluated and remains up-to-date. No changes have been necessary.

PRINTED FROM OXFORD MEDICINE ONLINE (www.oxfordmedicine.com). © Oxford University Press, 2021. All Rights Reserved. Under the terms of the licence agreement, an individual user may print out a PDF of a single chapter of a title in Oxford Medicine Online for personal use (for details see Privacy Policy and Legal Notice).

Science has always been part of Western medicine, although what counts as scientific has changed over the centuries, as have the content of medical knowledge, the tools of medical investigation, and the details of medical treatments. This brief overview develops a historical typology of medicine since antiquity. It divides the ‘kinds’ of medicine into five: bedside, library, hospital, social, and laboratory. These categories are still principal headings in modern health budgets, but they also have specific historical resonances. (1) Bedside medicine, developed by the Hippocratic doctors in classical times, has its modern counterpart in primary care. (2) Library medicine, associated with the scholastic mentality of the Middle Ages, still surfaces in the problems of information storage and retrieval in the computer age. (3) Hospital medicine, central to French medicine of the early 19th century, placed the diagnostic and therapeutic functions of the modern hospital centre stage in care and teaching. (4) Social medicine is about prevention, both communal and individual, and is especially visible in our notion of ‘lifestyle’ and its impact on health. (5) Laboratory medicine has its natural home in the research establishment and is a critical site for the creation of medical knowledge, setting the standards for both medical science and scientific medicine. François Magendie (1773–1855) was probably the first truly ‘modern’ medical scientist: he had little sense of medical tradition instead, he sought to establish medicine on new, scientific foundations.

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Prove that the Sorgenfrey line is totally disconnected

Let $ mathbb_l $ denote the topological space whose underlying set is the real line $ mathbb $ and the topology is generated by the half closed intervals $ [a,b) $. Prove that the topological space $ mathbb_l $ is totally disconnected.

A space is totally disconnected if its only connected components are one-point sets. Given any set $ Iin mathbb_l $, $ I=[a,b) $ for some $ ale b $ in $ R $. If $ a=b $, $ I $ is a one-point set and clearly a connected component (there are no $ A,Bin I $ both non-empty and proper). If $ a<b $, $ I $ is not a one-point set and there exists a $ c $ with $ a<c<b $. Then $ A=[a,c) $ and $ B=[c,b) $ are both non-empty, proper open subsets of $ I $ and they constitute a separation of $ I $ because $ Acap B=emptyset $ and $ Acup B=I $. It is therefore clear that the only connected components in $ mathbb_l $ are the one-point sets, and hence the topological space $ mathbb_l $ is totally disconnected.


Abstract

Objective

To determine the potential association of age-related macular degeneration (AMD), a representative chronic age-related degenerative disease of the retina associated with inflammation and aging, with susceptibility to SARS-CoV-2 infection and severe COVID-19 outcomes.

Design

Nationwide cohort study with propensity-score matching.

Setting

Population-based nationwide cohort in Korea.

Study population

Data were obtained from the Health Insurance Review & Assessment Service of Korea, including all patients older than 40 years who underwent SARS-CoV-2 testing in South Korea between January 1, 2020, and May 15, 2020 (excluding self-referral).

Main outcome measures

SARS-CoV-2 test positivity was the primary outcome and severe clinical outcome of COVID-19 was the secondary outcome.

Results

The unmatched cohort consisted of 135,435 patients who were tested for SARS-CoV-2 4531 (3.3%) tested positive for SARS-CoV-2 5493 (4.1%) patients had AMD. After propensity score matching, exudative AMD was associated with an increased likelihood of susceptibility to SARS-CoV-2 infection (adjusted odds ratio [aOR], 1.50 95% confidence interval [CI], 1.03-2.25), and a considerably greater risk of severe clinical outcomes of COVID-19 (aOR, 2.26 95% CI, 1.02 to 5.26), but not any AMD and non-exudative AMD.

Conclusions

In a Korean nationwide cohort, our data suggest that clinicians should be aware of the greater risk of susceptibility to severe clinical outcomes of COVID-19 in patients with exudative AMD. Our findings provide an improved understanding of the relationship between the pathogenesis of COVID-19 and chronic neurological disorders.


3. Relaxing Herbs To Improve Sleep Quality

There are several herbs that can help to improve sleep quality. I find that for someone with severe insomnia, these herbs don&rsquot quite do the job. In combination with other strategies, however, certain herbs can complement a sleep plan very well.

Some of my favorite herbs to improve sleep are kava, chamomile, valerian, passionflower, lavender, and lemon balm. Instead of going out and buying all of these herbs, I usually recommend this Nighty Night tea made by Traditional Medicinals that combines many of the best sleep herbs in one tea bag. Use this daily to improve sleep quality! We also offer a great supplement with clinical doses of these calming herbs called Relax Calm.


Materials and Methods

Construction of Loop assembly backbones

Loop assembly vectors were constructed using Gibson assembly (Gibson et al., 2009 ). Several changes were made to a pGreenII vector (Hellens et al., 2000 ) to obtain a basic plasmid backbone for the Loop assembly vectors: BsaI and SapI sites were removed from the plasmid using silent mutations when possible. In order to reduce issues with the stability of large constructs in bacteria (Moore et al., 2016 Watson et al., 2016 ), two nucleotides of the pGreenII ColEI-derived origin of replication were mutated, reversing it into the medium–low copy number pBR322 origin of replication. A region extending from the T-DNA left border to the hygromycin resistance gene cassette was replaced with the sequence of the pET15 vector (Haseloff, 1999 ) from the nptII nosT terminator to the UASGAL4 promoter (bases 2851–3527). A spectinomycin resistance was cloned to replace the nptI cassette to provide a microbial selection marker for the pEven plasmids. UNSs were cloned into the kanamycin and spectinomycin version of the vector backbones after the 3′ end of the pET15 vector sequence and the right border. Finally, the Loop restriction enzyme sites (BsaI and SapI), overhangs and the lacZα cassette were cloned in between the UNSs, yielding the pOdd and pEven vectors. L0 plasmids used for Loop Type IIS assembly were assembled using Gibson assembly into a modified pUDP2 (BBa_P10500) plasmid, which contained a 20-bp random sequence (5′-TAGCCGGTCGAGTGATACACTGAAGTCTC-3′) downstream of the 3′ convergent BsaI site and upstream of the BioBrick suffix, to provide nonhomologous flanking regions for correct orientation during overlap assembly.

DNA spacers

Random DNA sequences were retrieved from Random DNA Sequence Generator (https://www.faculty.ucr.edu/

mmaduro/random.htm), ordered as dsDNA fragments from IDT and assembled using Gibson assembly.

Plasmids and construct design

L0 parts used for DNA construction are described in Supporting Information Table S1 their sequences are included in the Supporting Information and are available through Addgene. Sequences for Loop plasmids and resulting multigene assemblies are included in Supporting Information.

The design of the constructs was performed using LoopDesigner software, installed on a local machine. The software was configured to use Loop assembly backbones together with BsaI and SapI REs, as well as A–B and α–ω overhangs. In addition, the definitions of 12 L0 part types were added to the software, based on the overhangs specified by the common syntax. The sequences of the L0 parts were added to the LoopDesigner database, assigning one of the defined part types, and assembled consequently into Level 1 and Level 2 constructs in silico. The concentrations of L0 parts and Level 1 constructs were adjusted to those suggested by LoopDesigner for 10-μl reactions.

Loop Type IIS assembly protocol

The Loop Type IIS assembly protocol was adapted from Patron ( 2016 ), and can be found at https://www.protocols.io/view/loop-assembly-pyqdpvw. An aliquot of 15 fmol of each part to be assembled was mixed with 7.5 fmol of the receiver plasmid in a final volume of 5 μl with distilled H2O (dH2O) (Table S2). The reaction mix, containing 3 μl of dH2O, 1 μl of T4 DNA ligase buffer 10× (no. B0202 NEB, Ipswich, MA, USA), 0.5 μl of 1 mg ml −1 purified bovine serum albumin (1 : 20 dilution in dH2O of BSA, Molecular Biology Grade 20 mg ml −1 , NEB cat. B9000), 0.25 μl of T4 DNA ligase at 400 U μl −1 (NEB cat. M0202) and 0.25 μl of corresponding restriction enzyme at 10 U μl −1 (BsaI NEB cat. R0535 or SapI NEB cat. R0569), was prepared on ice. Then, 5 μl of the reaction mix was combined with 5 μl of DNA mix for a reaction volume of 10 μl (Table S3) by pipetting, and incubated in a thermocycler using the program described in Table S4. For SapI reactions, T4 DNA ligase buffer was replaced by CutSmart buffer (NEB cat. B7204S) supplemented with 1 mM ATP 1 μl of the reaction mix was added to 50 μl of chemically competent TOP10 cells (no. C4040100 ThermoFisher) and, following incubation at 42°C for 30 s, samples were left on ice for 5 min, 250 μl of Super Optimal broth with Catabolite repression (SOC) medium was added and cells were incubated at 37°C for 1 h. Finally, 5 μl of 25 mg ml −1 of 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-Gal) (no. B4252 Sigma-Aldrich), dissolved in dimethylsulfoxide (DMSO), was added and the cells were plated onto selective Lysogeny broth (LB)-agar plates supplemented with 1 mM Isopropyl β- d -1-thiogalactopyranoside (IPTG) (no. I6758 Sigma-Aldrich). Assembly reactions were also automated. The assembly reactions were identical, but scaled down to a total volume of 1 μl. Reactions were set up on a Labcyte Echo (San Jose, CA, USA) in 384-well plates and incubated on a thermal cycling machine using the same conditions as described above. Reactions were transformed into 4 μl competent XL10-Gold ® Ultracompetent Cells (Agilent Technologies, Santa Clara, CA, USA) and plated onto eight-well selective LB-agar plates. Colonies were picked for growth in 1 ml of medium in 96-well plates on a Hamilton STARplus ® platform (Reno, NV, USA).

Standardized PCR of transcriptional units

PCR using UNS oligonucleotides was performed at an annealing temperature of 60°C, with 35 cycles using Phusion High-Fidelity DNA polymerase (no. F-530 ThermoFisher) in 50-μl reactions, according to the manufacturer's instructions. Template was added to a final concentration of 20 pg μl −1 . DNA fragments were visualized using SYBR Safe DNA Gel Stain (no. S33102 ThermoFisher) on a blue LED transilluminator (IORodeo, Pasadena, CA, USA). DNA purification was performed using a NucleoSpin Gel and PCR Clean-up purification kit (no. 740609.250 Macherey-Nagel, Düren, Germany). UNS primers used in TU amplification are listed in Table S5.

Validation by sequencing

The sequences of the assembled plasmids were verified by complete sequencing using 150-base pair paired-end reads on an Illumina MiSeq platform, and can be found in the EMBL-ENA database grouped under study PRJEB29863. Libraries were prepared using the Nextera XT DNA Library Prep Kit (no. FC-131-1096 Illumina Inc., San Diego, CA, USA), using the manufacturer's protocol modified to a one in four dilution. Reads were filtered and trimmed for low-quality bases and mapped to plasmids using the ‘map to reference tool’ from Geneious 8.1.8 software (https://www.geneious.com Kearse et al., 2012 ), with standard parameters. Sequence fidelity was determined manually.

Agrobacterium-mediated Marchantia transformation

Agrobacterium-mediated transformation was carried out as described previously (Ishizaki et al., 2008 ), with the following exceptions: half of an archegonia-bearing sporangium (spore-head) was used for each transformation. Dried spore-heads were crushed in a 50-ml Falcon tube with a 15-ml Falcon tube and resuspended in 1 ml of water per spore-head. Resuspended spores were filtered through a 40-μm mesh (no. 352340 Corning Inc., NY, USA) and 1 ml of suspension was aliquoted into a 1.5-ml Eppendorf tube and centrifuged at 13 000 g for 1 min at room temperature. The supernatant was discarded and spores were resuspended in 1 ml of sterilization solution, and incubated at room temperature for 20 min at 150 rpm on an orbital shaker. The sterilization solution was prepared by dissolving one Milton mini-sterilizing tablet (Milton Pharmaceutical UK, Cheltenham, UK active ingredient, sodium dichloroisocyanurate CAS: 2893-78-9: 19.5% w/w) in 25 ml of sterile water. Samples were centrifuged at 13 000 g for 1 min, washed once with sterile water and resuspended in 100 μl of sterile water per spore-head used. One hundred microlitres of sterilized spores were inoculated onto half-strength Gamborg's B5 1% (w/v) agar plates and grown under constant fluorescent lighting (50–60 mol photons m −2 s −1 ) upside down for 5 d until co-cultivation. Sporelings were co-cultivated with previously transformed and induced Agrobacterium GV2260 transformed with the pSoup plasmid (Hellens et al., 2000 ) in 250-ml flasks containing 25 ml of half-strength Gamborg's B5 medium supplemented with 5% (w/v) sucrose, 0.1% (w/v) N-Z Amine A (Sigma cat. C7290), 0.03% (w/v) L-glutamine (Sigma cat. G8540) and 100 μM acetosyringone (Sigma-Aldrich cat. D134406) for 36 h, until washing and plating onto selective medium.

Laser scanning confocal microscopy

A microscope slide was fitted with a 65-μl Gene Frame (ThermoFisher cat. AB0577) and 65 μl of dH2O was placed in the centre. Marchantia gemmae was carefully deposited on the drop of dH2O using a small inoculation loop and a #0 coverslip was attached to the Gene Frame. Slides were examined on a Leica, Wetzlar, Germany TCS SP8 confocal microscope platform equipped with a white-light laser (WLL) device. Imaging was conducted using a Leica HC PL APO 20× CS2 air objective with a sequential scanning mode with laser wavelengths of 405, 488 and 515 nm, capturing emitted fluorescence at 450–482-, 492–512- and 520–550-nm windows, respectively, in each sequential scan. Z-stacks were collected every 5 μm for the complete volume range and maximum intensity projections were processed using Image J software. Fluorescence bleedthrough from the blue pseudocoloured channel (membrane-localized enhanced green fluorescent protein (eGFP)) into the green pseudocoloured channel (nuclear-localized Venus) was eliminated using custom Python scripts which subtracted 20% of the value of pixels present in the blue channel to the green channel. Images were edited to scale the pixel intensity to the full 8-bit range and a merged image was processed.

Transient expression in Arabidopsis mesophyll protoplasts

Well-expanded leaves from 3–4-wk-old Arabidopsis plants (Columbia-0) were used for protoplast transfection. Plants were grown at 22°C in low-light (75 μmol m −2 s −1 ) and short-photoperiod (12 h : 12 h, light : dark) conditions. Protoplasts were isolated and polyethylene glycol (PEG) transfected according to Yoo et al. ( 2007 ). For transfection, 6 μl of Loop L2 plasmids (2 μg μl −1 ), isolated by a NucleoBond Xtra Midi/Maxi purification kit (Macherey-Nagel cat. 740410.50), were used. Transfected protoplasts were incubated for 12 h in light and then visualized by epifluorescent microscopy in a Neubauer chamber (Hirschmann Laborgeräte, Eberstadt, Germany).

Epifluorescence microscopy

Transfected protoplasts were visualized using a Nikon Ni microscope (Minato, Tokyo, Japan) equipped with 49021 ET – EBFP2/Coumarin/Attenuated DAPI (excitation, 405/20 nm dichroic, 425 nm emission, 460/50 nm), 96227 AT-EYFP (excitation, 495/20 nm dichroic, 515 nm emission, 540/30 nm), 96223 AT-ECFP/C (excitation, 495/20 nm dichroic, 515 nm emission, 540/30 nm) and 96312 G-2E/C (excitation, 540/20 nm dichroic, 565 nm emission, 620/60 nm) filter cubes.

LoopDesigner

In order to implement an object-oriented model for Loop assembly, we built a PartsDB library (https://github.com/HaseloffLab/PartsDB) to define several interlinked classes, each of which is associated with a table in a relational SQL database. The structure of LoopDesigner is built around a Part class, which either represents an ordered collection of children parts from which it is assembled, or a DNA sequence in the case of L0 parts. In this way, we ensured that the actual DNA sequence is only stored once, and the sequences of L1 and higher parts are constructed on demand from the relational links. In addition, each Part is associated with one of the Backbone instances which, together with a Part sequence, represents a complete Loop assembly plasmid. Every instance of a Backbone class is a combination of a Base Sequence and a donor Restriction Enzyme Site, for example, pOdd 1-4 and pEven 1-4 are Backbone instances in the schema described in this article. Base Sequence represents a type of receiver plasmid, for example, pOdd and pEven, and is composed of a DNA sequence of the plasmid and an instance of a receiver Restriction Enzyme Site. Finally, Restriction Enzyme Site class is composed of a Restriction Enzyme instance, which stores the restriction enzyme recognition sequence, and a pair of overhang sequences, which can be either receiver or donor overhangs.


Open Research

Age measurements from the plasma proteomic dataset derived from 4263 individuals (aged 18–95 years) are accessible via an online software tool (https://twc-stanford.shinyapps.io/aging_plasma_proteome/). The full plasma proteomic dataset derived from 3301 individuals (aged 18–76 years) is available in the European Genotype Archive (accession number EGAS00001002555).

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Watch the video: The Complement System HD Animation (May 2022).


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