Prospective Memory Training in Older Adults: A Systematic Review and Meta-Analysis.

Prospective memory (PM), which enables one to remember to carry out delayed intentions, is crucial for everyday functioning. PM commonly deteriorates upon cognitive decline in older adults, but several studies have shown that PM in older adults can be improved by training. The current study aimed to summarise this evidence by conducting a qualitative systematic analysis and quantitative meta-analysis of the effects of PM training in older adults, for which systematic searches were conducted across seven databases (Cochrane Library, Embase, PubMed, PsycInfo, Web of Science, CINAHL and Scopus). Forty-eight studies were included in the qualitative analysis, and 43% of the assessed PM training interventions showed positive gains in enhancing PM. However, the methodological quality varied across the studies, with 41% of the non-randomised control trials (non-RCTs) rated as having either serious or critical risk of bias. Therefore, only 29 RCTs were included in the subsequent quantitative meta-analysis. We found a significant and moderate immediate efficacy (Hedges' g = 0.54) of PM training in enhancing PM performance in older adults, but no significant long-term efficacy (Hedges' g = 0.20). Two subgroup analyses also revealed a robust training efficacy across the study population (i.e., healthy and clinical population) and the number of training sessions (i.e., single session and programme-based). Overall, this study provided positive evidence to support PM training in older adults. Further studies are warranted to explore the mechanisms by which PM training exerts its effects, and better-quality RCTs are needed to provide more robust evidence supporting our findings.

Risk of Postpolypectomy Bleeding With Uninterrupted Clopidogrel Therapy in an Industry-Independent, Double-Blind, Randomized Trial.

BACKGROUND & AIMS: Guidelines recommend withholding clopidogrel 7 days before polypectomy to decrease bleeding risk, but these were written based on limited evidence. We investigated whether uninterrupted clopidogrel therapy increases the risk of delayed postpolypectomy bleeding in patients undergoing colonoscopy. METHODS: We identified patients receiving clopidogrel for cardiovascular disease undergoing elective colonoscopies in Hong Kong from February 28, 2012 through April 11, 2018. Eligible patients were instructed to stop taking clopidogrel 7 days before colonoscopy. Then, they were randomly assigned to groups given clopidogrel (75 mg) or placebo daily until the morning of colonoscopy. All patients resumed their usual prescriptions of clopidogrel after colonoscopy. The primary end point was delayed postpolypectomy bleeding that required hospitalization or intervention up to 30 days after colonoscopy. Secondary end points were immediate postpolypectomy bleeding and serious cardio-thrombotic events for as long as 6 months after colonoscopy, according to Antithrombotic Trialists' criteria. All events were adjudicated by an independent masked committee. RESULTS: In total, 387 patients underwent colonoscopy and 216 required polypectomies (106 patients in the clopidogrel group and 110 patients in the placebo group). The cumulative incidence of delayed postpolypectomy bleeding was 3.8% (95% confidence interval 1.4-9.7) in the clopidogrel group and 3.6% (95% confidence interval 1.4-9.4) in the placebo group (P = .945 by log-rank test). There were no significant differences in immediate postpolypectomy bleeding (8.5% vs 5.5%; P = .380) and cardio-thrombotic events (1.5% vs 2%; P = .713). CONCLUSIONS: In a randomized controlled trial of clopidogrel users undergoing colonoscopy, a slightly larger proportion of patients continuing clopidogrel developed delayed and immediate postpolypectomy bleeding, although this difference was not statistically significant. ClinicalTrials.gov, number NCT01806090.

Microbial Community Composition and Putative Biogeochemical Functions in the Sediment and Water of Tropical Granite Quarry Lakes.

Re-naturalized quarry lakes are important ecosystems, which support complex communities of flora and fauna. Microorganisms associated with sediment and water form the lowest trophic level in these ecosystems and drive biogeochemical cycles. A direct comparison of microbial taxa in water and sediment microbial communities is lacking, which limits our understanding of the dominant functions that are carried out by the water and sediment microbial communities in quarry lakes. In this study, using the 16S rDNA amplicon sequencing approach, we compared microbial communities in the water and sediment in two re-naturalized quarry lakes in Singapore and elucidated putative functions of the sediment and water microbial communities in driving major biogeochemical processes. The richness and diversity of microbial communities in sediments of the quarry lakes were higher than those in the water. The composition of the microbial communities in the sediments from the two quarries was highly similar to one another, while those in the water differed greatly. Although the microbial communities of the sediment and water samples shared some common members, a large number of microbial taxa (at the phylum and genus levels) were prevalent either in sediment or water alone. Our results provide valuable insights into the prevalent biogeochemical processes carried out by water and sediment microbial communities in tropical granite quarry lakes, highlighting distinct microbial processes in water and sediment that contribute to the natural purification of the resident water.

Microorganisms meet solid minerals: interactions and biotechnological applications.

In natural and engineered environments, microorganisms often co-exist and interact with various minerals or mineral-containing solids. Microorganism-mineral interactions contribute significantly to environmental processes, including biogeochemical cycles in natural ecosystems and biodeterioration of materials in engineered environments. In this mini-review, we provide a summary of several key mechanisms involved in microorganism-mineral interactions, including the following: (i) solid minerals serve as substrata for biofilm development; (ii) solid minerals serve as an electron source or sink for microbial respiration; (iii) solid minerals provide microorganisms with macro or micronutrients for cell growth; and (iv) (semi)conductive solid minerals serve as extracellular electron conduits facilitating cell-to-cell interactions. We also highlight recent developments in harnessing microbe-mineral interactions for biotechnological applications.

The role of micronutrients and strategies for optimized continual glycerol production from carbon dioxide by Dunaliella tertiolecta.

The microalga Dunaliella tertiolecta synthesizes intracellular glycerol as an osmoticum to counteract external osmotic pressure in high saline environments. The species has recently been found to release and accumulate extracellular glycerol, making it a suitable candidate for sustainable industrial glycerol production if a sufficiently high product titre yield can be achieved. While macronutrients such as nitrogen and phosphorus are essential and well understood, this study seeks to understand the influence of the micronutrient profile on glycerol production. The effects of metallic elements calcium, magnesium, manganese, zinc, cobalt, copper, and iron, as well as boron, on glycerol production as well as cell growth were quantified. The relationship between cell density and glycerol productivity was also determined. Statistically, manganese recorded the highest improvement in glycerol production as well as cell growth. Further experiments showed that manganese availability was associated with higher superoxide dismutase formation, thus suggesting that glycerol production is negatively affected by oxidative stress and the manganese bound form of this enzyme is required in order to counteract reactive oxygen species in the cells. A minimum concentration of 8.25 × 10(-5) g L(-1) manganese was sufficient to overcome this problem and achieve 10 g L(-1) extracellular glycerol, compared to 4 g L(-1) without the addition of manganese. Unlike cell growth, extracellular glycerol production was found to be negatively affected by the amount of calcium present in the normal growth medium, most likely due to the lower cell permeability at high calcium concentrations. The inhibitory effects of iron also affected extracellular glycerol production more significantly than cell growth and several antagonistic interaction effects between various micronutrients were observed. This study indicates how the optimization of these small amounts of nutrients in a two-stage system can lead to a large enhancement in D. tertiolecta glycerol production and should be considered during the design of a large scale bioprocess for this alternative route to glycerol.

Does the PedsQL reflect the real-time quality of life in autistic adolescents? A comparison with the experience sampling methodology.

BACKGROUND: The PedsQL is widely used to retrospectively evaluate quality of life (QoL) in autistic adolescents. However, concerns have arisen regarding its ability to reflect real-time QoL, considering their challenges in recollecting past experiences. OBJECTIVE: We examined the overall and social QoLs of autistic adolescents compared to neurotypical peers using the PedsQL and the experience sampling method (ESM), an ecological momentary assessment of QoL in real-time. Additionally, we explored the relationship between these measures in each group. METHODS: A total of 175 participants, including 117 autistic and 58 neurotypical adolescents aged 10-16, completed the PedsQL and an ESM protocol with a mobile device to record their real-time experiences seven times a day for seven days. We performed multiple linear regression and multilevel analyses to compare QoLs between groups and the association between the two measures. RESULTS: Autistic adolescents scored significantly lower than neurotypical peers on PedsQL overall and social QoL but not on the real-time experiences collected with ESM. Among neurotypical adolescents, we found significant associations between the Social Functioning score of the PedsQL and various aspects of real-time social experiences recorded with ESM. For autistic adolescents, only the real-time experience of 'loneliness' during social engagement was associated with Social Functioning on the PedsQL. CONCLUSIONS: The retrospective PedsQL does not entirely capture the real-time QoL via ESM. However, relying solely on ESM may overlook situations where participants opt out or could not complete surveys. Thus, using both retrospective and real-time assessments to examine QoL among autistic adolescents is recommended.

Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

The impact of coping behaviors on perceived competence and social anxiety in the everyday social engagement of autistic adolescents.

LAY ABSTRACT: Individuals who cope well with challenges may engage in social situations more successfully. We examined how well autistic adolescents coped, depending on how competent they felt and how much anxiety they experienced during social activities. We included 133 individuals (82 autistic, 51 neurotypical) between the ages of 10 and 16 years. Participants carried a mobile device that prompted them seven times a day for 7 days to record what they were doing, how competent they felt and how much anxiety they experienced. We used the Coping Inventory to understand how well participants coped with environmental challenges and met their needs for growth. Autistic adolescents were more likely than neurotypical peers to feel anxious while doing activities with adults. Autistic adolescents who had more difficulty coping with challenges were more likely to feel anxious when doing leisure activities with peers. Interestingly, autistic adolescents who coped better with challenges tended to feel less competent in social situations. However, those better able to meet their needs for growth tended to perceive their social competence positively. These findings can help practitioners develop strategies and programs to reduce the negative social experiences of autistic adolescents by helping them cope better.

A Population-Based Cohort Study of the Association between Visual Loss and Risk of Suicide and Mental Illness in Taiwan.

The psychosocial and health consequences of ocular conditions that cause visual impairment (VI) are extensive and include impaired daily activities, social isolation, cognitive impairment, impaired functional status and functional decline, increased reliance on others, increased risk of motor vehicle accidents, falls and fractures, poor self-rated health, and depression. We aimed to determine whether VI increases the likelihood of a poor prognosis, including mental illness, suicide, and mortality over time. In this large, location, population-based, nested, cohort study, we used data from 2000 to 2015 in the Taiwan National Health Insurance Research Database (NHIRD), which includes diagnoses of all the patients with VI. Baseline features, comorbidities, and prognostic variables were evaluated using a 1:4-matched cohort analysis. Furthermore, comparisons were performed using Cox regression and Bonferroni-correction (for multiple comparisons) to study the association between VI and poor prognosis (mental illness, suicide). The study outcome was the cumulative incidence of poor prognosis among the visually impaired and controls. A two-tailed Bonferroni-corrected p < 0.001 was considered statistically significant. Among the 1,949,101 patients enlisted in the NHIRD, 271 had been diagnosed with VI. Risk factors for poor prognosis and the crude hazard ratio was 3.004 (95% confidence interval 2.135-4.121, p < 0.001). Participants with VI had an increased risk of poor prognosis according to the sensitivity analysis, with a poor prognosis within the first year and first five years. VI was associated with suicide and mental health risks. This study revealed that patients with VI have a nearly 3-fold higher risk of psychiatric disorders, including anxiety, depression, bipolar, and sleep disorders, than the general population. Early detection through comprehensive examinations based on increased awareness in the clinical context may help maintain visual function and avoid additional complications.

The Dark Side of Microbial Processes: Accumulation of Nitrate During Storage of Surface Water in the Dark and the Underlying Mechanism.

In densely populated cities with limited land, storage of surface water in underground spaces is a potential solution to meet the rising demand of clean water. In addition, due to the imperative need of renewable solar energy and limited land resources, the deployment of floating solar photovoltaic (PV) systems over water has risen exponentially. In both scenarios, microbial communities in the water do not have access to sunlight. How the absence of sunlight influences microbial community function and the water quality is largely unknown. The objective of this study was to reveal microbial processes in surface water stored in the dark and water quality dynamics. Water from a freshwater reservoir was stored in the dark or light (control) for 6 months. Water quality was monitored at regular intervals. RNA sequencing was performed on the Illumina MiSeq platform and qPCR was used to substantiate the findings arising from the sequencing data. Our results showed that storage of surface water in the dark resulted in the accumulation of nitrate in the water. Storage in the dark promoted the decay of algal cells, increasing the amount of free nitrogen in the water. Most of the free nitrogen was eventually transformed into nitrate through microbial processes. RNA sequencing-based microbial community analyses and pure culture experiments using nitrifying bacteria Nitrosomonas europaea and Nitrobacter sp. revealed that the accumulation of nitrate in the dark was likely due to an increase in nitrification rate and a decrease in the assimilation rate of nitrate back into the biomass. IMPORTANCE Microbial communities play an essential role in maintaining a healthy aquatic ecosystem. For example, in surface water reservoirs, microorganisms produce oxygen, break down toxic contaminants and remove excess nitrogen. In densely populated cities with limited land, storing surface water in underground spaces and deploying floating solar photovoltaic (PV) systems over water are potential solutions to address water and energy sustainability challenges. In both scenarios, surface water is kept in the dark. In this work, we revealed how the absence of sunlight influences microbial community function and water quality. We showed that storage of surface water in the dark affected bacterial activities responsible for nitrogen transformation, resulting in the accumulation of nitrate in the water. Our findings highlight the importance of monitoring nitrate closely if raw surface water is to be stored in the dark and the potential need of downstream treatment to remove nitrate.

Nisin-inducible secretion of a biologically active single-chain insulin analog by Lactococcus lactis NZ9000.

Oral delivery of insulin to diabetic patients is highly desirable because it would be non-invasive and more closely mimic normal physiology, but this route of administration typically results in low bioavailability due to low pH and enzymatic degradation along the gastrointestinal tract. To explore an alternative approach that may mitigate these obstacles and also facilitate local synthesis of new therapeutic protein molecules in the small intestine, we engineered the food-grade bacterium Lactococcus lactis (NZ9000) for nisin-inducible expression and secretion of a bioactive single-chain insulin (SCI) analog, SCI-57. We show that the addition of nisin during early-log phase has a modest inhibitory effect on cell growth but induction during mid-log phase has a negligible impact on proliferation, suggesting a tradeoff between cell growth rate and duration of induction. We find that a signal peptide such as usp45 is necessary for secretion of SCI-57 into the medium; furthermore, we demonstrate that this secreted SCI-57 is biologically active, as assessed by the ability of conditioned L. lactis medium to stimulate Akt signaling in differentiated 3T3-L1 adipocytes. Finally, we show that the biological activity of SCI-57 was enhanced by near-neutral or slightly alkaline pH during induction, which is comparable to the pH in the small intestine, and by removal of a C-terminal purification tag. This study demonstrates that food-grade bacteria can be engineered to secrete bioactive insulin analogs and opens up the possibility of oral insulin delivery using live microorganisms.

Effects of overexpression of X-box binding protein 1 on recombinant protein production in Chinese hamster ovary and NS0 myeloma cells.

X-box binding protein 1 (XBP-1) is a key regulator of the cellular secretory pathway and unfolded protein response (UPR). It has been shown that the spliced form of XBP-1, XBP-1S, functions as a transcription activator and up-regulates many genes associated with protein secretion and biosynthesis of endoplasmic reticula. Since the production of some recombinant proteins is widely believed to be limited by the secretory capacity of the host cell, an increase in protein production may be achieved by overexpressing XBP-1S. In this study, the effects of XBP-1S on the productivity of monoclonal antibody (MAb), interferon gamma (IFNgamma), and erythropoietin (EPO) are examined in Chinese hamster ovary (CHO) and NS0 cell lines. Results show that XBP-1S may become a determinative factor only when accumulation of recombinant proteins exceeds the secretory capacity of the host cell. In transient transfection systems where a bottleneck in protein secretion was achieved, overexpression of XBP-1S improved protein titers by up to 2.5-fold. In contrast, overexpression of XBP-1S had no detectable effects on protein productivity of stable cell lines that did not exhibit any secretory bottleneck. We conclude that overexpression of XBP-1S is an effective strategy in enhancing recombinant protein production when the secretory pathway of the host cell is saturated by high-level synthesis of recombinant proteins.

Regulation of the NRF2 transcription factor by andrographolide and organic extracts from plant endophytes.

The transcription factor NF-E2 Related Factor-2 (NRF2) is an important drug target. Activation of NRF2 has chemopreventive effects in cancer and exerts beneficial effects in a number of diseases, including neurodegenerative diseases, inflammatory diseases, hepatosteatosis, obesity and insulin resistance. Hence, there have been great efforts to discover and characterize novel NRF2 activators. One reported NRF2 activator is the labdane diterpenoid andrographolide. In this study, we identified the mechanism through which andrographolide activates NRF2. We showed that andrographolide inhibits the function of KEAP1, a protein that together with CUL3 and RBX1 forms an E3 ubiquitin ligase that polyubiquitinates NRF2. Andrographolide partially inhibits the interaction of KEAP1 with CUL3 in a manner dependent on Cys151 in KEAP1. This suggests that andrographolide forms Michael acceptor dependent adducts with Cys151 in KEAP1 in vivo, leading to inhibition of NRF2 ubiquitination and consequently accumulation of the transcription factor. Interestingly, we also showed that at higher concentrations andrographolide increases NRF2 protein expression in a Cys151 independent, but likely KEAP1 dependent manner, possibly through modification of other Cys residues in KEAP1. In this study we also screened secondary metabolites produced by endophytes isolated from non-flowering plants for NRF2-inducing properties. One of the extracts, ORX 41, increased both NRF2 protein expression and transcriptional activity markedly. These results suggest that endophytes isolated from non-flowering or other plants may be a good source of novel NRF2 inducing compounds.

NP-Sticky: a web server for optimizing DNA ligation with non-palindromic sticky ends.

High-efficiency DNA ligation is vital for many molecular biology experiments, and it is best achieved using reactants with non-palindromic sticky ends to maximize specificity. However, optimizing such multi-parametric ligation reactions often involves extensive trial and error. We have developed a freely available Web-based ligation calculator, NP-Sticky (http://sarkarlab.umn.edu/npsticky/), that predicts product distribution for given reactant concentrations, thus enabling straightforward computational optimization of these reactions. Built-in schemes include two-piece and three-piece linear ligation, as well as insert-vector circular ligation. The only parameters needed for the underlying thermodynamic model are the free energies of ligation for each sticky end, which can be estimated by the calculator from the overhang sequences or provided by the user from direct experimental measurement. Free energies of sticky-end mismatches are also calculated for determining the extent of byproduct formation. This ligation calculator allows rapid identification of the optimal conditions for maximizing incorporation, efficiency, and/or accuracy, based on specific needs.

Intracellular glycerol accumulation in light-limited Dunaliella tertiolecta culture is determined by partitioning of glycerol across the cell membrane.

Dunaliella accumulates intracellular glycerol to counterbalance the extracellular salinity. In N-limited chemostat cultures of D. tertiolecta, total glycerol production (sum of intracellular and extracellular) and intracellular glycerol content were proportional to the salinity of the culture medium. In the light-limited D. tertiolecta culture, total glycerol output (sum of intracellular and extracellular) was relatively constant at different salinities (0.5 and 2.0 M), while the intracellular glycerol content was proportional to the culture medium salinity, that is, the cells released less glycerol into the culture medium, rather than de novo synthesis of glycerol at high culture medium salinity. The study implies different regulatory mechanisms in the accumulation of intracellular glycerol in N-limited and light-limited D. tertiolecta in response to salinity.

Streamlined protocol for mRNA display.

mRNA display is a powerful method for in vitro directed evolution of polypeptides, but its time-consuming, technically demanding nature has hindered its widespread use. We present a streamlined protocol in which lengthy mRNA purification steps are replaced with faster precipitation and ultrafiltration alternatives; additionally, other purification steps are entirely eliminated by using a reconstituted translation system and by performing reverse transcription after selection, which also protects input polypeptides from thermal denaturation. We tested this procedure by performing affinity selection against Her2 using binary libraries containing a nonspecific designed ankyrin repeat protein (DARPin) doped with a Her2-binding DARPin (dopant fraction ranging from 1:10 to 1:10 000). The Her2-binding DARPin was recovered in all cases, with an enrichment factor of up to 2 orders of magnitude per selection round. The time required for 1 round is reduced from ∼4-7 days to 2 days with our protocol, thus simplifying and accelerating mRNA display experiments.

Effective phagocytosis of low Her2 tumor cell lines with engineered, aglycosylated IgG displaying high FcγRIIa affinity and selectivity.

Glycans anchored to residue N297 of the antibody IgG Fc domain are critical in mediating binding toward FcγRs to direct both adaptive and innate immune responses. However, using a full length bacterial IgG display system, we have isolated aglycosylated Fc domains with mutations that confer up to a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous human inhibitory receptor, FcγRIIb. The mutant Fc domain (AglycoT-Fc1004) contained a total of 5 amino acid substitutions that conferred an activating to inhibitory ratio of 25 (A/I ratio; FcyRIIa-R131:FcγRIIb). Incorporation of this engineered Fc into trastuzumab, an anti-Her2 antibody, resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with both medium and low Her2-expressing cancer cells. A mathematical model has been developed to help explain how receptor affinity and the A/I ratio relate to improved antibody dependent cell-mediated phagocytosis. Our model provides guidelines for the future engineering of Fc domains with enhanced effector function.

Continual production of glycerol from carbon dioxide by Dunaliella tertiolecta.

Microalgae have high photosynthetic efficiencies and produce many valuable compounds from carbon dioxide. The Dunaliella genus accumulates glycerol, yet no commercial process currently exists for glycerol production from this microalga. Here it was found that in addition to intracellular accumulation, Dunaliella tertiolecta also releases glycerol into the external medium continuously, forming a large and stable carbon pool. The process is not affected by nutrient starvation or onset of cell death. Carbon dioxide was fixed at a constant rate, the bulk of it being channelled to extracellular glycerol (82%), resulting in enhanced photosynthetic carbon assimilation of 5 times that used for biomass production. The final extracellular glycerol concentration was 34 times the maximum concentration of intracellular glycerol; the latter declined further during cell death. Findings from this work will assist in the development of a bioconversion process to produce glycerol using D. tertiolecta without the need for cell harvest or disruption.

Model-guided ligation strategy for optimal assembly of DNA libraries.

DNA ligation is essential to many molecular biology manipulations, but this reaction is often carried out by following generic guidelines or by trial and error. Maximizing the desired ligation product is especially important in DNA library construction for directed evolution experiments since library diversity is directly affected by ligation efficiency. Here, we suggest that display vectors that rely on Type IIP restriction sites for cloning should be redesigned to utilize Type IIS restriction sites instead because ligation yield is significantly improved: we observed up to 15- and 2.6-fold increases in desired products for circular and linear ligation reactions, respectively. To guide ligation optimization more rationally, we developed an easily parameterized thermodynamic model that predicts product distributions based on input DNA concentrations and free energies of the ligation events. We applied this model to study ligation reactions using a ribosome display vector redesigned with Type IIS restriction sites (pRDV2). We computationally predicted and experimentally validated the relative abundance of various products in three-piece linear ligations as well as the extent of transformation from vector-insert circular ligations. Based on our results, we provide general insights into ligation and we outline guidelines for optimizing this reaction for both in vivo and in vitro display methodologies.