Influence of Red Blood Cells on Channel Characterization in Cylindrical Vasculature.

Molecular communication via diffusion (MCvD) expects Brownian motions of the information molecules to transmit information. However, the signal propagation largely depends on the geometric characteristics of the assumed flow model, i.e., the characteristics of the environment, design, and position of the transmitter and receiver, respectively. These characteristics are assumed to be lucid in many ways by either consideration of one-dimensional diffusion, unbounded environment, or constant drift. In reality, diffusion often occurs in blood-vessel-like channels. To this aim, we try to study the effect of the biological environment on channel performance. The Red-Blood Cells (RBCs) found in blood vessels enforces a higher concentration of molecules towards the vessel walls, leading to better reception. Therefore, in this paper we derive an analytical expression of Channel Impulse Response (CIR) for a dispersion-advection-based regime, contemplating the influence of RBCs in the model and considering a point source transmitter and a realistic design of the receiver.

Metformin ameliorates neuroinflammatory environment for neurons and astrocytes during in vitro and in vivo stroke and tobacco smoke chemical exposure: Role of Nrf2 activation.

Despite the protective nature of the blood-brain barrier (BBB) and brain-protecting tissues, some types of CNS injury or stress can cause cerebral cytokine production and profound alterations in brain function. Neuroinflammation, which can also be accompanied by increased cerebral cytokine production, has a remarkable impact on the pathogenesis of many neurological illnesses, including loss of BBB integrity and ischemic stroke, yet effective treatment choices for these diseases are currently lacking. Although little is known about the brain effects of Metformin (MF), a commonly prescribed first-line antidiabetic drug, prior research suggested that it may be useful in preventing BBB deterioration and the increased risk of stroke caused by tobacco smoking (TS). Therefore, reducing neuroinflammation by escalating anti-inflammatory cytokine production and declining pro-inflammatory cytokine production could prove an effective therapeutic strategy for ischemic stroke. Hence, the current investigation was planned to explore the potential role of MF against stroke and TS-induced neuroinflammation and reactive oxygen species (ROS) production. Our studies revealed that MF suppressed releasing pro-inflammatory mediators like tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) by aiming at the nuclear factor kappa B (NF-κB) signaling pathway in primary neurons and astrocytes. MF also upregulated anti-inflammatory mediators, like interleukin-10 (IL-10), and interleukin-4 (IL-4), by upregulating the Nrf2-ARE signaling pathway. Adolescent mice receiving MF along with TS exposure also showed a notable decrease in NF-κB expression compared to the mice not treated with MF and significantly decreased the level of TNF-α, IL-1ß, MCP-1, and MIP-2 and increased the levels of IL-10 and IL-4 through the activation of Nrf2-ARE signaling pathway. These results suggest that MF has anti-neuroinflammatory effects via inhibiting NF-κB signaling by activating Nrf2-ARE. These studies support that MF could be a strong candidate drug for treating and or preventing TS-induced neuroinflammation and ischemic stroke.

Multidisciplinary advances in kombucha fermentation, health efficacy, and market evolution.

Kombucha, a fermented tea beverage, has seen a significant rise in global popularity. This increase is attributed to its reported health benefits and extensive cultural heritage. The comprehensive review examines kombucha through microbiology, biochemistry, and health sciences, highlighting its therapeutic potential and commercial viability. Central to kombucha production is the symbiotic culture of bacteria and yeasts (SCOBY), which regulates a complex fermentation process, resulting in a bioactive-rich elixir. The study examines the microbial dynamics of SCOBY, emphasizing the roles of various microorganisms. It focuses the contributions of acetic acid bacteria, lactic acid bacteria, and osmophilic yeasts, including genera such as Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Brettanomyces/Dekkera, and Pichia. These microorganisms play crucial roles in producing bioactive compounds, including organic acids, polyphenols, and vitamins. These bioactive compounds confer therapeutic properties to kombucha. These properties include antioxidant, antimicrobial, anti-inflammatory, antidiabetic, antihypertensive, cancer prevention, hepatoprotective, and detoxifying effects. The review also explores the growing market for kombucha, driven by consumer demand for functional beverages and opportunities for innovative product development. It emphasizes the necessity of standardized production to ensure safety and validate health claims. Identifying research gaps, the review highlights the importance of clinical trials to verify therapeutic benefits. Ultimately, this study integrates traditional knowledge with scientific research, providing directions for future studies and commercial expansion, emphasizing the role of kombucha in health and wellness.

Atrial Septal Defect Stent Compression During Resuscitation of an Infant With Hypoplastic Left Heart Syndrome.

A newborn with hypoplastic left heart syndrome underwent a Norwood procedure with a Sano shunt. A month later, he had an atrial septal defect (ASD) stent placed due to ASD size and flow restriction. Three weeks later, he had sudden bradycardia and cardiac arrest in the intensive care unit. He was resuscitated and cannulated for extracorporeal membrane oxygenation (ECMO) through a right neck approach. An attempt to wean him from ECMO a week later failed due to hypoxia. Echocardiography showed ASD flow restriction and a chest x-ray showed flattening of the left atrial side of the ASD stent from chest compression resulting in a restrictive ASD. The patient underwent surgical removal of the ASD stent with ASD enlargement and was successfully weaned from ECMO after surgery.

A Review: Surface Engineering of Lipid-Based Drug Delivery Systems.

This review explores the evolution of lipid-based nanoparticles (LBNPs) for drug delivery (DD). Herein, LBNPs are classified into liposomes and cell membrane-based nanoparticles (CMNPs), each with unique advantages and challenges. Conventional LBNPs possess drawbacks such as poor targeting, quick clearance, and limited biocompatibility. One of the possible alternatives to overcome these challenges is surface modification of nanoparticles (NPs) with materials such as polyethylene glycol (PEG), aptamers, antibody fragments, peptides, CD44, hyaluronic acid, folic acid, palmitic acid, and lactoferrin. Thus, the main focus of this review will be on the different surface modifications that enable LBNPs to have beneficial properties for DD, such as enhancing mass transport properties, immune evasion, improved stability, and targeting. Moreover, various CMNPs are explored used for DD derived from cells such as red blood cells (RBCs), platelets, leukocytes, cancer cells, and stem cells, highlighting their unique natural properties (e.g., biocompatibility and ability to evade the immune system). This discussion extends to the biomimicking of hybrid NPs accomplished through the surface coating of synthetic (mainly polymeric) NPs with different cell membranes. This review aims to provide a comprehensive resource for researchers on recent advances in the field of surface modification of LBNPs and CMNPs. Overall, this review provides valuable insights into the dynamic field of lipid-based DD systems.

In vitro Models for Predicting Bioadhesion Fracture Strength to Ex Vivo Animal Buccal Tissue.

Commitment to the 3Rs principle (Replacement, Reduction, and Refinement) led to the development of a cell-based system to measure buccal bioadhesion in vitro and replace the use of porcine buccal and esophageal tissues (PBT and PET, respectively). Additionally, the aim is to bridge the gap in knowledge regarding the bioadhesion properties of PBT and PET. The in vitro models are based on the human buccal epithelial cell line-TR146 without ("Model I") or with ("Model II") 5% (w/v) mucous layer. The in vitro setup also provides a method to evaluate the bioadhesion between two soft materials. Standard bioadhesive hydrogels (alginate, chitosan, and gelatin) are used to test and compare the results from the in vitro models to the ex vivo tissues. The ex vivo and in vitro models show increased bioadhesion as the applied force and contact time increases. Furthermore, Model I exhibits bioadhesion values-of alginate, chitosan, and gelatin-comparable to those obtained with PBT. It is also found that contact time and applied force similarly affect PBT and PET bioadhesion, while PET exhibits greater values. In conclusion, Model I can replace PBT for measuring bioadhesion and be incorporated into the experimental design of bioadhesive DDS, thus minimizing animal tissue usage.

Evaluating the accuracy of a state-of-the-art large language model for prediction of admissions from the emergency room.

BACKGROUND: Artificial intelligence (AI) and large language models (LLMs) can play a critical role in emergency room operations by augmenting decision-making about patient admission. However, there are no studies for LLMs using real-world data and scenarios, in comparison to and being informed by traditional supervised machine learning (ML) models. We evaluated the performance of GPT-4 for predicting patient admissions from emergency department (ED) visits. We compared performance to traditional ML models both naively and when informed by few-shot examples and/or numerical probabilities. METHODS: We conducted a retrospective study using electronic health records across 7 NYC hospitals. We trained Bio-Clinical-BERT and XGBoost (XGB) models on unstructured and structured data, respectively, and created an ensemble model reflecting ML performance. We then assessed GPT-4 capabilities in many scenarios: through Zero-shot, Few-shot with and without retrieval-augmented generation (RAG), and with and without ML numerical probabilities. RESULTS: The Ensemble ML model achieved an area under the receiver operating characteristic curve (AUC) of 0.88, an area under the precision-recall curve (AUPRC) of 0.72 and an accuracy of 82.9%. The naïve GPT-4's performance (0.79 AUC, 0.48 AUPRC, and 77.5% accuracy) showed substantial improvement when given limited, relevant data to learn from (ie, RAG) and underlying ML probabilities (0.87 AUC, 0.71 AUPRC, and 83.1% accuracy). Interestingly, RAG alone boosted performance to near peak levels (0.82 AUC, 0.56 AUPRC, and 81.3% accuracy). CONCLUSIONS: The naïve LLM had limited performance but showed significant improvement in predicting ED admissions when supplemented with real-world examples to learn from, particularly through RAG, and/or numerical probabilities from traditional ML models. Its peak performance, although slightly lower than the pure ML model, is noteworthy given its potential for providing reasoning behind predictions. Further refinement of LLMs with real-world data is necessary for successful integration as decision-support tools in care settings.

Bacteriophages as a potential substitute for antibiotics: A comprehensive review.

Over the years, the administration of antibiotics for the purpose of addressing bacterial infections has become increasingly challenging due to the increased prevalence of antimicrobial resistance exhibited by various strains of bacteria. Multidrug-resistant (MDR) bacterial species are rising due to the unavailability of novel antibiotics, leading to higher mortality rates. With these conditions, there is a need for alternatives in which phage therapy has made promising results. Phage-derived endolysins, phage cocktails, and bioengineered phages are effective and have antimicrobial properties against MDR and extensively drug-resistant strains. Despite these, it has been observed that phages can give antimicrobial activity to more than one bacterial species. Thus, phage cocktail against resistant strains provides broad spectrum treatment and magnitude of effectivity, which is many folds higher than antibiotics. Many commercially available endolysins such as Staphefekt SA.100, Exebacase (CF-301), and N-Rephasin®SAL200 are used in biofilm penetration and treating plant diseases. The role of CMP1 phage endolysin in transgenic tomato plants in preventing Clavibacter michiganensis infection and the effectiveness of phage in protecting Atlantic salmon from vibriosis have been reported. Furthermore, phage-derived endolysin therapy, such as TSPphg phage exogenous treatment, can aid in disrupting cell walls, leading to bacterial cell lysis. As animals in aquaculture and slaughterhouses are highly susceptible to bacterial infections, effective phage therapy instead of antibiotics can help treat poultry animals, preserve them, and facilitate disease-free trade. Using bioengineered phages and phage cocktails enhances the effectiveness by providing a broad spectrum of phages and target specificity. Research is currently being conducted on clinical trials to confirm the efficacy of engineered phages and phage cocktails in humans. Although obtaining commercial approval may be time-consuming, it will be beneficial in the postantibiotic era. This review provides an overview of the significance of phage therapy as a potential alternative to antibiotics in combating resistant bacterial strains and its application to various fields and emphasizes the importance of safeguarding and ensuring treatment efficacy.

Physical function patient-reported outcomes among adolescent and young adult cancer survivors: A systematic review.

BACKGROUND: The physical challenges faced by adolescents and young adults (AYA) after a cancer diagnosis may be different from those experienced by paediatric and older adult cancer patients. Patient-reported outcome measures (PROMs) are valuable tools that can be useful in exploring the experiences of AYAs and identifying important issues, recurrent themes and areas to potentially improve quality of life. OBJECTIVE: We compared patient-reported physical function outcomes between AYAs diagnosed with cancer and non-cancer controls. METHOD: This paper builds on a scoping review published in early 2023 and focuses on PROMs related to physical function. RESULTS: This systematic review includes 16 studies that measured and reported on physical function PROMs in AYA cancer survivors compared with their cancer-free peers. Of these studies, 14 found that physical function in AYA survivors was significantly worse. This paper also includes a meta-analysis conducted on 5 studies using the EORTC-QLQ-C30 to measure physical function, which found that physical function score was an average of 7.03 (95% CI: -10.21, -3.86) points lower in the AYA cancer group, compared to their cancer free-peers, a difference that is clinically meaningful. CONCLUSIONS: The results overwhelmingly demonstrate that AYAs post a cancer diagnosis have worse health-related quality of life from a physical function perspective than their cancer-free peers, providing a compelling argument for the need to address this issue. All but one of the studies were cross-sectional, which highlights the need for further assessment of this group longitudinally throughout their cancer journey.

Evaluation of the Existence of Post-COVID-19 Tachycardia in a Community Healthcare System.

Background: Post-coronavirus disease 2019 (COVID-19) syndrome derives from lingering symptoms after an acute COVID-19 infection. Palpitation was one of the most common symptoms of post-COVID-19 syndrome that correlated with objective data such as persisting sinus tachycardia; but to our best knowledge, there is a scarcity of research regarding the association of COVID-19 and sinus tachycardia in the post-acute setting. Therefore, the purpose was to identify if there is an association between COVID-19 infection and sinus tachycardia in the post-acute phase, namely post-COVID-19 tachycardia (PCT) other than inappropriate sinus tachycardia (IST) and postural orthostatic tachycardia syndrome (POTS). Methods: This retrospective observational study entails 1,425 patients admitted for COVID-19 infection with the interest in finding an association with PCT. The prevalence of PCT was evaluated using descriptive statistics, predictions of patient characteristics and comorbidities were identified using multinomial logistic regression, and associations between patient comorbidities and characteristics were evaluated with corresponding Pearson Chi-square test and post hoc tests Phi and Cramer's V. Results: The percentage of patients with PCT in our sample of interest was an average of 28.18%. There was a strong association of PCT with patients of age group less than 65 years. Other clinical characteristics, such as shorter length of stay, unknown smoking status, and patients with commercial type insurance, had significant association with PCT. COVID-19 severity categorized as "less severe", readmission rates within 30 days, and patients with less comorbidities were more likely to be associated with PCT. Conclusions: PCT is likely a separate entity from IST and POTS, and an important entity under the umbrella of post-COVID-19 syndrome. It warrants further studies to elucidate the underlying pathophysiology and to confirm its presence as a distinct entity.

Algae-Based Nanoparticles for Oral Drug Delivery Systems.

Drug administration by oral delivery is the preferred route, regardless of some remaining challenges, such as short resident time and toxicity issues. One strategy to overcome these barriers is utilizing mucoadhesive vectors that can increase intestinal resident time and systemic uptake. In this study, biomimetic nanoparticles (NPs) were produced from 14 types of edible algae and evaluated for usage as oral DDSs by measuring their size, surface charge, morphology, encapsulation efficiency, mucoadhesion force, and cellular uptake into Caco-2 cells. The NPs composed of algal materials (aNPs) exhibited a spherical morphology with a size range of 126-606 nm and a surface charge of -9 to -38 mV. The mucoadhesive forces tested ex vivo against mice, pigs, and sheep intestines revealed significant variation between algae and animal models. Notably, Arthospira platensis (i.e., Spirulina) NPs (126 ± 2 nm, -38 ± 3 mV) consistently exhibited the highest mucoadhesive forces (up to 3127 ± 272 µN/mm²). Moreover, a correlation was found between high mucoadhesive force and high cellular uptake into Caco-2 cells, further supporting the potential of aNPs by indicating their ability to facilitate drug absorption into the human intestinal epithelium. The results presented herein serve as a proof of concept for the possibility of aNPs as oral drug delivery vehicles.

Patient Reported Fatigue Among Adolescent and Young Adult Cancer Patients Compared to Non-Cancer Patients: A Systematic Review and Meta-Analysis.

Adolescent and young adult (AYA) cancer patients and survivors are a growing population due to more frequent diagnoses and improved survival. Fatigue is a common symptom experienced by cancer patients and it is often missed by health care professionals. Patient reported outcome measures (PROMs) can assist in evaluating patient reported fatigue. This systematic review aims to determine if AYA cancer patients report more fatigue than AYAs who have not been diagnosed with cancer. We used a subset of articles from a larger review that searched PubMed, EMBASE, CINAHL, and PsycINFO to determine which PROMs and domains are currently being used to evaluate AYA cancer. This study identified 175 articles related to PROMs in the AYA cancer population. Articles with PROMs reporting on fatigue/vitality were used in this review. From the original 175 articles, we identified 8 fatigue/vitality articles for this review. All eight articles found an increase in fatigue/decrease in vitality in the AYA cancer population compared to healthy controls. A meta-analysis was performed on four articles that used the same PROM tool (EORTC QLQ-C30). This found a statistically significant and clinically meaningful increase in mean fatigue of 12.5 95% confidence interval: 3.3-21.8 points (scale 0-100, higher number indicates more fatigue) in the AYA cancer group compared to healthy noncancer controls. Fatigue in the AYA cancer population is a significant issue, it is often undetected and underreported, and early interventions are needed to prevent the negative subsequent sequelae.

Procalcitonin Use for Predicting Mortality and Morbidity of Patients Diagnosed With Sepsis Within the Intensive Care Unit.

Objective Infections leading to severe sepsis and septic shock are among the top five causes requiring admission to the intensive care unit (ICU). Up to 40% of ICU admissions contain a sepsis diagnosis. Without a clear marker to diagnose and manage sepsis, procalcitonin has been extensively studied for its usefulness in the management of bacterial infections. These studies, however, have been focused toward how it can be used to help guide when antibiotics should be initiated and de-escalated. There, however, has not been a study on how this biomarker could be used to predict mortality, and morbidity and help guide a need for antibiotic escalation. Design A retrospective chart review was conducted on patients admitted to the ICU at Northeast Georgia Medical Center between January 1, 2019, to June 30, 2021. Inclusion criteria were all patients above the age of 18 admitted to the ICU with a diagnosis of sepsis and having at least two procalcitonin drawn within 10 days of each other. Exclusion criteria were any patient with a diagnosis of COVID-19. Data Analysis was conducted to identify how delta procalcitonin could identify mortality and morbidity and if there was any change in antibiotics based on the delta procalcitonin.  Conclusion There was a statistically significant association between a delta positive procalcitonin and increased ICU length of stay. There was no statistical significance in expiration based on the antibiotic change in relationship to delta positive change in procalcitonin.

Aortic Thrombosis and Subsequent Myocardial Infarction in a Previously Healthy 12-Year-Old Male.

Aortic thrombus formation in children is uncommon, particularly in an otherwise healthy pediatric patient. Thromboembolism of such thrombi resulting in subsequent ST-segment elevation myocardial infarction is, thus, exceedingly rare. (Level of Difficulty: Intermediate.).

A Paradigm Shift from International to Transnational Medical Education.

In recent years, the number of medical students seeking international opportunities has grown. To satisfy these demands, collaborative international programs have been developed. However, the benefits of these programs are limited as they employ an international medical education (IME) approach where only the students are exchanged. In this commentary, we discuss the current models of IME and propose a paradigm shift to a transnational approach wherein the student, faculty, and curriculum are exchanged allowing for increased integration and awareness of cultural and educational approaches to treatment that can be retained and incorporated into future practice to advance healthcare across the globe.

Targeting allosteric binding site in methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to identify natural product inhibitors via structure-based computational approach.

Cancer has been viewed as one of the deadliest diseases worldwide. Among various types of cancer, breast cancer is the most common type of cancer in women. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a promising druggable target and is overexpressed in cancerous cells, like, breast cancer. We conducted structure-based modeling on the allosteric site of the enzyme. Targeting the allosteric site avoids the problem of drug resistance. Pharmacophore modeling, molecular docking, HYDE assessment, drug-likeness, ADMET predictions, simulations, and free-energy calculations were performed. The RMSD, RMSF, RoG, SASA, and Hydrogen-bonding studies showed that seven candidates displayed stable behaviour. As per the literature, average superimposed simulated structures revealed a similar protein conformational change in the αE'-ßf' loop, causing its displacement away from the allosteric site. The MM-PBSA showed tight binding of six compounds with the allosteric pocket. The effect of inhibitors interacting in the allosteric site causes a decrease in the binding energy of J49 (active-site inhibitor), suggesting the effect of allosteric binding. The PCA and FEL analysis revealed the significance of the docked compounds in the stable behaviour of the complexes. The outcome can contribute to the development of potential natural products with drug-like properties that can inhibit the MTHFD2 enzyme.

Advancing edema detection: Harnessing the power of machine learning and near infrared spectroscopy for cerebral and cerebellar edema assessment.

Edema, characterized by brain swelling, is a common response observed in various brain injuries. Timely detection of edema is crucial to mitigate the associated risks and improve patient care. This study evaluates the efficacy of CEREBO®, a non-invasive machine learning-powered near-infrared spectroscopy (mNIRS) based device, in detecting edema. The study was conducted on 234 participants with suspected head injuries who underwent simultaneous CEREBO® scans and CT head scans. The results of the study showed that CEREBO® effectively identified edematous lobes, achieving a sensitivity of 95.7%, specificity of 97%, and accuracy of 96.9% for cases with intracranial hemorrhage (ICH). Additionally, for cases without ICH, the device exhibited a sensitivity of 100%, specificity of 97.2%, and accuracy of 97.2%. Two cases were reported where CEREBO® failed to detect edematous ICH. The study highlights the potential of CEREBO® as a valuable tool for early detection of pre-symptomatic edema and ICH, enabling timely interventions and improved patient care. The findings support the reliability of near-infrared spectroscopy as a diagnostic modality for edema.

Safety and immunogenicity of a new formulation of a pentavalent DTwP-HepB-Hib vaccine in healthy Indian infants-A randomized study.

BACKGROUND: Pentavalent vaccines (DTP-HepB-Hib) have been introduced in many countries in their routine public immunization programmes to protect against diphtheria (D), tetanus (T), pertussis (P), hepatitis B (Hep B) and Hemophilus influenzae type b (Hib) diseases. This study compared the safety and immunogenicity of a new formulation of a whole-cell Bordetella pertussis (wP) based pentavalent vaccine (DTwP-HepB-Hib). The new formulation was developed using well-characterized hepatitis B and pertussis whole cell vaccine components. METHODS: This was a phase III, observer-blind, randomized, non-inferiority, multi-center study conducted in India among 460 infants who were followed up for safety and immunogenicity for 28 days after administration of three doses of either investigational or licensed comparator formulations at 6-8, 10-12 and 14-16 weeks of age. RESULTS: The investigational formulation of DTwP-HepB-Hib vaccine was non-inferior to the licensed formulation in terms of hepatitis B seroprotection rate (% of subjects with HepB antibodies ≥10mIU/mL were 99.1% versus 99.0%, respectively, corresponding to a difference of 0.1% (95% CI, -2.47 to 2.68)) and pertussis immune responses (adjusted geometric mean concentrations of antibodies for anti-PT were 76.7 EU/mL versus 63.3 EU/mL, with a ratio of aGMTs of 1.21 (95% CI, 0.89-1.64), and for anti-FIM were 1079 EU/mL versus 1129 EU/mL, with a ratio of aGMTs of 0.95 (95% CI, 0.73-1.24), respectively). The immune responses to other valences (D, T, and Hib) in the two formulations were also similar. The safety profile of both formulations was found to be similar and were well tolerated. CONCLUSIONS: The investigational DTwP-HepB-Hib vaccine formulation was immunogenic and well-tolerated when administered as three dose primary series in infants. CLINICAL TRIAL REGISTRATION: Clinical Trials Registry India number: CTRI/2018/12/016692.

An expanded GCaMP reporter toolkit for functional imaging in Caenorhabditis elegans.

In living organisms, changes in calcium flux are integral to many different cellular functions and are especially critical for the activity of neurons and myocytes. Genetically encoded calcium indicators (GECIs) have been popular tools for reporting changes in calcium levels in vivo. In particular, GCaMPs, derived from GFP, are the most widely used GECIs and have become an invaluable toolkit for neurophysiological studies. Recently, new variants of GCaMP, which offer a greater variety of temporal dynamics and improved brightness, have been developed. However, these variants are not readily available to the Caenorhabditis elegans research community. This work reports a set of GCaMP6 and jGCaMP7 reporters optimized for C. elegans studies. Our toolkit provides reporters with improved dynamic range, varied kinetics, and targeted subcellular localizations. Besides optimized routine uses, this set of reporters is also well suited for studies requiring fast imaging speeds and low magnification or low-cost platforms.

High-throughput library transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS).

High-throughput transgenesis using synthetic DNA libraries is a powerful method for systematically exploring genetic function. Diverse synthesized libraries have been used for protein engineering, identification of protein-protein interactions, characterization of promoter libraries, developmental and evolutionary lineage tracking, and various other exploratory assays. However, the need for library transgenesis has effectively restricted these approaches to single-cell models. Here, we present Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS), a simple yet powerful approach to large-scale transgenesis that overcomes typical limitations encountered in multicellular systems. TARDIS splits the transgenesis process into a two-step process: creation of individuals carrying experimentally introduced sequence libraries, followed by inducible extraction and integration of individual sequences/library components from the larger library cassette into engineered genomic sites. Thus, transformation of a single individual, followed by lineage expansion and functional transgenesis, gives rise to thousands of genetically unique transgenic individuals. We demonstrate the power of this system using engineered, split selectable TARDIS sites in Caenorhabditis elegans to generate (1) a large set of individually barcoded lineages and (2) transcriptional reporter lines from predefined promoter libraries. We find that this approach increases transformation yields up to approximately 1000-fold over current single-step methods. While we demonstrate the utility of TARDIS using C. elegans, in principle the process is adaptable to any system where experimentally generated genomic loci landing pads and diverse, heritable DNA elements can be generated.

Transgenesis ­ the ability to insert foreign genetic material (known as transgenes) in to the genome of an organism ­ has revolutionized biological research. This approach has made it possible for scientists to study the role of specific genes and to produce animal models which mimic aspects of human diseases. For transgenes to be maintained and passed down to future generations, they must be introduced into germ cells which will go on to form the egg and sperm of the organism. However, despite advances in genetic engineering, this process (called 'specific transgenesis') is still laborious and time-consuming, and limits researchers to working with only a small number of known DNA sequences at a time. In contrast, 'exploratory transgenesis' ­ where dozens of transgenes from a library of DNA sequences are introduced simultaneously into multiple individuals ­ is more efficient and allows for more large-scale experiments. However, this approach can only be done with single-celled organisms like bacteria, and remains virtually impossible in laboratory animals like worms or mice. Stevenson et al. therefore set out to boost the efficiency of exploratory transgenesis in a commonly used laboratory animal, the roundworm Caenorhabditis elegans. To do this, they used the 'library' principle of exploratory transgenesis in order to develop a new resource called TARDIS (short for, Transgenic Arrays Resulting in Diversity of Integrated Sequences). First, Stevenson et al. genetically engineered worms to carry a 'landing site' for foreign DNA. Next, a library of transgenes and a mechanism which cuts pieces of DNA and pastes them into the landing site were introduced into the germ cells of these worms using traditional methods. The worms were then bred to generate a large population of offspring that had inherited this array of foreign DNA sequences. Finally, the 'cut and paste' mechanism was switched on and a random transgene was inserted into the landing site in the genome. This resulted in thousands of worms which each had a unique genetic modification that can be passed on to future generations. These results show for the first time that larger-scale transgenesis experiments are possible in multi-cellular animals. In the future, Stevenson et al. hope that TARDIS can be adapted to different organisms and allow researchers to carry out experiments that were not previously possible.