Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

Tensor-based insights into systems immunity and infectious disease.

Profiling immune responses across several dimensions, including time, patients, molecular features, and tissue sites, can deepen our understanding of immunity as an integrated system. These studies require new analytical approaches to realize their full potential. We highlight recent applications of tensor methods and discuss several future opportunities.

The gut-brain and gut-macrophage contribution to gastrointestinal dysfunction with systemic inflammation.

The gastrointestinal tract is one of the main organs affected during systemic inflammation and disrupted gastrointestinal motility is a major clinical manifestation. Many studies have investigated the involvement of neuroimmune interactions in regulating colonic motility during localized colonic inflammation, i.e., colitis. However, little is known about how the enteric nervous system and intestinal macrophages contribute to dysregulated motility during systemic inflammation. Given that systemic inflammation commonly results from the innate immune response against bacterial infection, we mimicked bacterial infection by administering lipopolysaccharide (LPS) to rats and assessed colonic motility using ex vivo video imaging techniques. We utilized the Cx3cr1-Dtr rat model of transient depletion of macrophages to investigate the role of intestinal macrophages in regulating colonic motility during LPS infection. To investigate the role of inhibitory enteric neurotransmission on colonic motility following LPS, we applied the nitric oxide synthase inhibitor, Nω-nitro-L-arginine (NOLA). Our results confirmed an increase in colonic contraction frequency during LPS-induced systemic inflammation. However, neither the depletion of intestinal macrophages, nor the suppression of inhibitory enteric nervous system activity impacted colonic motility disruption during inflammation. This implies that the interplay between the enteric nervous system and intestinal macrophages is nuanced, and complex, and further investigation is needed to clarify their joint roles in colonic motility.

Tofacitinib and peficitinib inhibitors of Janus kinase for autoimmune disease treatment: a quantum biochemistry approach.

Autoimmune inflammatory diseases, such as rheumatoid arthritis (RA) and ulcerative colitis, are associated with an uncontrolled production of cytokines leading to the pronounced inflammatory response of these disorders. Their therapy is currently focused on the inhibition of cytokine receptors, such as the Janus kinase (JAK) protein family. Tofacitinib and peficitinib are JAK inhibitors that have been recently approved to treat rheumatoid arthritis. In this study, an in-depth analysis was carried out through quantum biochemistry to understand the interactions involved in the complexes formed by JAK1 and tofacitinib or peficitinib. Computational analyses provided new insights into the binding mechanisms between tofacitinib or peficitinib and JAK1. The essential amino acid residues that support the complex are also identified and reported. Additionally, we report new interactions, such as van der Waals; hydrogen bonds; and alkyl, pi-alkyl, and pi-sulfur forces, that stabilize the complexes. The computational results revealed that peficitinib presents a similar affinity to JAK1 compared to tofacitinib based on their interaction energies.

Disulfide-HMGB1 signals through TLR4 and TLR9 to induce inflammatory macrophages capable of innate-adaptive crosstalk in human liver transplantation.

Ischemia-reperfusion injury (IRI) during orthotopic liver transplantation (OLT) contributes to graft rejection and poor clinical outcomes. The disulfide form of high mobility group box 1 (diS-HMGB1), an intracellular protein released during OLT-IRI, induces pro-inflammatory macrophages. How diS-HMGB1 differentiates human monocytes into macrophages capable of activating adaptive immunity remains unknown. We investigated if diS-HMGB1 binds toll-like receptor (TLR) 4 and TLR9 to differentiate monocytes into pro-inflammatory macrophages that activate adaptive immunity and promote graft injury and dysfunction. Assessment of 106 clinical liver tissue and longitudinal blood samples revealed that OLT recipients were more likely to experience IRI and graft dysfunction with increased diS-HMGB1 released during reperfusion. Increased diS-HMGB1 concentration also correlated with TLR4/TLR9 activation, polarization of monocytes into pro-inflammatory macrophages, and production of anti-donor antibodies. In vitro, healthy volunteer monocytes stimulated with purified diS-HMGB1 had increased inflammatory cytokine secretion, antigen presentation machinery, and reactive oxygen species production. TLR4 inhibition primarily impeded cytokine/chemokine and costimulatory molecule programs, whereas TLR9 inhibition decreased HLA-DR and reactive oxygen species production. diS-HMGB1-polarized macrophages also showed increased capacity to present antigens and activate T memory cells. In murine OLT, diS-HMGB1 treatment potentiated ischemia-reperfusion-mediated hepatocellular injury, accompanied by increased serum alanine transaminase levels. This translational study identifies the diS-HMGB1/TLR4/TLR9 axis as potential therapeutic targets in OLT-IRI recipients.

medna-metadata: an open-source data management system for tracking environmental DNA samples and metadata.

MOTIVATION: Environmental DNA (eDNA), as a rapidly expanding research field, stands to benefit from shared resources including sampling protocols, study designs, discovered sequences, and taxonomic assignments to sequences. High-quality community shareable eDNA resources rely heavily on comprehensive metadata documentation that captures the complex workflows covering field sampling, molecular biology lab work, and bioinformatic analyses. There are limited sources that provide documentation of database development on comprehensive metadata for eDNA and these workflows and no open-source software. RESULTS: We present medna-metadata, an open-source, modular system that aligns with Findable, Accessible, Interoperable, and Reusable guiding principles that support scholarly data reuse and the database and application development of a standardized metadata collection structure that encapsulates critical aspects of field data collection, wet lab processing, and bioinformatic analysis. Medna-metadata is showcased with metabarcoding data from the Gulf of Maine (Polinski et al., 2019). AVAILABILITY AND IMPLEMENTATION: The source code of the medna-metadata web application is hosted on GitHub (https://github.com/Maine-eDNA/medna-metadata). Medna-metadata is a docker-compose installable package. Documentation can be found at https://medna-metadata.readthedocs.io/en/latest/?badge=latest. The application is implemented in Python, PostgreSQL and PostGIS, RabbitMQ, and NGINX, with all major browsers supported. A demo can be found at https://demo.metadata.maine-edna.org/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Examining enteric nervous system function in rat and mouse: an interspecies comparison of colonic motility.

Gastrointestinal motility is crucial to gut health and has been associated with different disorders such as inflammatory bowel diseases and postoperative ileus. Despite rat and mouse being the two animal models most widely used in gastrointestinal research, minimal studies in rats have investigated gastrointestinal motility. Therefore, our study provides a comparison of colonic motility in the mouse and rat to clarify species differences and assess the relative effectiveness of each animal model for colonic motility research. We describe the protocol modifications and optimization undertaken to enable video imaging of colonic motility in the rat. Apart from the broad difference in terms of gastrointestinal diameter and length, we identified differences in the fundamental histology of the proximal colon such that the rat had larger villus height-to-width and villus height-to-crypt depth ratios compared with mouse. Since gut motility is tightly regulated by the enteric nervous system (ENS), we investigated how colonic contractile activity within each rodent species responds to modulation of the ENS inhibitory neuronal network. Here we used Nω-nitro-l-arginine (l-NNA), an inhibitor of nitric oxide synthase (NOS) to assess proximal colon responses to the stimulatory effect of blocking the major inhibitory neurotransmitter, nitric oxide (NO). In rats, the frequency of proximal colonic contractions increased in the presence of l-NNA (vs. control levels) to a greater extent than in mice. This is despite a similar number of NOS-expressing neurons in the myenteric plexus across species. Given this increase in colonic contraction frequency, the rat represents another relevant animal model for investigating how gastrointestinal motility is regulated by the inhibitory neuronal network of the ENS.NEW & NOTEWORTHY Mice and rats are widely used in gastrointestinal research but have fundamental differences that make them important as different models for different questions. We found that mice have a higher villi length-to-width and villi length-to-crypt depth ratio than rat in proximal colon. Using the ex vivo video imaging technique, we observed that rat colon has more prominent response to blockade of major inhibitory neurotransmitter (nitric oxide) in myenteric plexus than mouse colon.

Optical refrigeration of payloads to T < 125†K.

A modified all-solid-state optical cryocooler prototype based on anti-Stokes fluorescence in a 10%-doped Yb:YLF crystal cooled a payload to temperatures below 125 K starting from room temperature. To achieve this record performance, the optical refrigerator employed a novel, to the best of our knowledge, textured-MgF2 thermal link to improve the thermal transport and fluorescence escape. Additionally, it used spectrally selective, high-reflection coatings in the pump circulator cavity to suppress parasitic lasing and amplified spontaneous emission.

Melon genotypes with resistance to Liriomyza sativae Blanchard (Diptera: Agromyzidae).

The vegetable leaf miner (Liriomyza sativae) is considered one of the main melon pests, causing serious problems for producers in all growing regions. A promising type of pest control has been use of resistant cultivars, in isolation or associated with other types of control. This study aimed to evaluate the resistance of melon genotypes to L. sativae. Twenty-one melon genotypes and one commercial "Goldex" hybrid (susceptibility pattern) were evaluated in two experiments. In the first experiment, we observed the non-preference of L. sativae for oviposition and feeding by quantifying the number of eggs and feeding punctures, both on the adaxial side and on the abaxial face of the leaves. In the second experiment, we observed the antibiosis effect through L. sativae larval and pupal viability. Genotype CNPH 06-1047-341 showed the lowest preference for oviposition (high resistance), with low egg values on both leaf sides (0.3 eggs/plant). In genotypes CNPH 06-1047-313, CNPH 06-1047-346, CNPH 11-1071-27, CNPH 11-1071-39, CNPH 11-1071-43, and CNPH 11-1071-53, we observed a higher preference for the adaxial side, whereas for the other genotypes and the commercial hybrid there was no discrimination between leaf sides. In relation to antibiosis, genotypes CNPH 06-1047-339, CNPH 06-1047-333, CNPH 06-1047-330, CNPH 06-1047-334, CNPH 06-1047-331, CNPH 06-1047-343, CNPH 10-1056-313, CNPH 06-1047-346, and CNPH 06-1047-341 presented lower larval and pupal viability. Genotype CNPH 06-1047-341 was the least preferred for oviposition and feeding and the most promising as a source of resistance to L. sativae.

Kinase inhibitor screening reveals aurora-a kinase is a potential therapeutic and prognostic biomarker of gastric cancer.

Gastric cancer is one of the most common and deadly types of cancer in the world, and poor prognosis with treatment failure is widely reported in the literature. In this context, kinases have been considered a relevant choice for targeted therapy in gastric cancer. Here, we explore the antiproliferative and antimigratory effects of the AURKA inhibitor and the prognostic and therapeutic value as a biomarker of gastric cancer. A total of 145 kinase inhibitors were screened to evaluate the cytotoxic or cytostatic effects in the gastric cancer cell line. Using the Alamar Blue assay, flow cytometry, quantitative polymerase chain reaction, and observation of caspase 3/7 activity and cell migration, we investigated the antiproliferative, proapoptotic, and antimigratory effects of the AURKA inhibitor. Moreover, AURKA overexpression was evaluated in the gastric cell lines and the gastric tumor tissue. Out of the 145 inhibitors, two presented the highest antiproliferative effect. Both molecules can induce apoptosis by the caspases 3/7 pathway in addition to inhibiting cancer cell migration, mainly the AURKA inhibitor. Moreover, molecular docking analysis revealed that GW779439X interacts in the active site of the AURKA enzyme with similar energy as a well-described inhibitor. Our study identified AURKA overexpression in the gastric cancer cell line and gastric tumor tissue, revealing that its overexpression in patients with cancer is correlated with low survival. Therefore, it is feasible to suggest AURKA as a potential marker of gastric cancer, besides providing robust information for diagnosis and estimated survival of patients. AURKA can be considered a new molecular target used in the prognosis and therapy of gastric cancer.

Long-term cancer surveillance: results from the Forteo Patient Registry Surveillance Study.

The Forteo Patient Registry estimated the incidence of osteosarcoma in US patients treated with teriparatide and enrolled in the study between 2009 and 2019. No incident cases of osteosarcoma were identified among patients registered, and the crude incidence rate was 0 (95% confidence interval [CI], 0-10.2) cases per million person-years. PURPOSE: The prospective, voluntary Forteo Patient Registry was established to estimate the incidence of osteosarcoma in patients who have received treatment with teriparatide (Forteo). METHODS: Information on US adults prescribed teriparatide and enrolled in the Forteo Patient Registry 2009-2019 was linked with data from participating state cancer registries annually (2010-2019) to identify incident osteosarcoma cases using a standardized linkage algorithm. Teriparatide exposure was ascertained from self-reported data that included teriparatide initiation and demographics necessary to complete linkage. Osteosarcoma cases diagnosed on or after January 1, 2009, were identified by participating state cancer registries. The crude incidence rate (IR) and standardized incidence ratio (SIR) of observed cases to the expected number of cases adjusted to the background rate (3 per million person-years) and corresponding 95% CIs for the occurrence of osteosarcoma were calculated whereby the cumulative amount of person-time observed was adjusted for mortality. RESULTS: Data for 75,247 enrolled patients (representing 361,763 cumulative person-years) were linked to each of 42 participating state cancer registries (covering 93% of the US population), which included information on 6180 cases of osteosarcoma. No matches with incident cases of osteosarcoma following registry enrollment were found. The crude IR was 0 (95% CI, 0-10.2) cases per million person-years and the SIR was 0 (95% CI, 0-3.0). CONCLUSIONS: The ability to draw conclusions about the incidence of osteosarcoma among patients participating in the registry was limited due to the smaller than expected amount of patient follow-up time and the fact that no cases were identified.

AGS3 and Gαi3 Are Concomitantly Upregulated as Part of the Spindle Orientation Complex during Differentiation of Human Neural Progenitor Cells.

Adult neurogenesis is modulated by many Gi-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gαi subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (ß-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gαi3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gαi3 in ENStem-A cells, and this specificity for Gαi3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gαi subunits. Moreover, the binding of Gαi3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gαi3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis.

The Effects of Green Tea Amino Acid L-Theanine Consumption on the Ability to Manage Stress and Anxiety Levels: a Systematic Review.

The green tea amino acid, L-theanine (L-THE) is associated with several health benefits, including improvements in mood, cognition and a reduction of stress and anxiety-like symptoms. This systematic review evaluated the effect of pure L-THE intake, in the form of orally administered nutritional supplements, on stress responses and anxiety levels in human randomised controlled trials. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist, 9 peer-reviewed journal articles were identified where L-THE as a supplement was compared to a control. Our findings suggest that supplementation of 200-400 mg/day of L-THE may assist in the reduction of stress and anxiety in people exposed to stressful conditions. Despite this finding, longer-term and larger cohort clinical studies, including those where L-THE is incorporated into the diet regularly, are needed to clinically justify the use of L-THE as a therapeutic agent to reduce stress and anxiety in people exposed to stressful conditions.

Respiration resolved imaging with continuous stable state 2D acquisition using linear frequency SWEEP.

PURPOSE: To investigate the potential of continuous radiofrequency (RF) shifting (SWEEP) as a technique for creating densely sampled data while maintaining a stable signal state for dynamic imaging. METHODS: We present a method where a continuous stable state of magnetization is swept smoothly across the anatomy of interest, creating an efficient approach to dense multiple 2D slice imaging. This is achieved by introducing a linear frequency offset to successive RF pulses shifting the excited slice by a fraction of the slice thickness with each successive repeat times (TR). Simulations and in vivo imaging were performed to assess how this affects the measured signal. Free breathing, respiration resolved 4D volumes in fetal/placental imaging is explored as potential application of this method. RESULTS: The SWEEP method maintained a stable signal state over a full acquisition reducing artifacts from unstable magnetization. Simulations demonstrated that the effects of SWEEP on slice profiles was of the same order as that produced by physiological motion observed with conventional methods. Respiration resolved 4D data acquired with this method shows reduced respiration artifacts and resilience to non-rigid and non-cyclic motion. CONCLUSIONS: The SWEEP method is presented as a technique for improved acquisition efficiency of densely sampled short-TR 2D sequences. Using conventional slice excitation the number of RF pulses required to enter a true steady state is excessively high when using short-TR 2D acquisitions, SWEEP circumvents this limitation by creating a stable signal state that is preserved between slices.

Conservation genomics in the fight to help the recovery of the critically endangered Siamese crocodile Crocodylus siamensis.

Endangered species are often characterized by low genetic diversity and it is imperative for conservation efforts to incorporate the knowledge obtained from genetic studies for effective management. However, despite the promise of technological advances in sequencing, application of genome-wide data to endangered populations remains uncommon. In the present study we pursued a holistic conservation-genomic approach to inform a field-based management programme of a Critically Endangered species, the Siamese crocodile Crocodylus siamensis. Using thousands of single nucleotide polymorphisms from throughout the genome, we revealed signals of introgression from two other crocodile species within our sample of both wild and captive-bred Siamese crocodiles from Cambodia. Our genetic screening of the Siamese crocodiles resulted in the subsequent re-introduction of 12 individuals into the wild as well as the selection of four individuals for captive breeding programmes. Comparison of intraspecific genetic diversity revealed an alarmingly low contemporary effective population size in the wild (<50) with evidence of a recent bottleneck around Tonle Sap Lake. We also projected a probable future extinction in the wild (within fewer than five generations) in this population in the absence of re-introduction efforts. However, an increase in the number of potential breeders through re-introductions, including the one resulting from this project, could counter this trend. Our results have been implemented in ongoing re-introduction and captive breeding programmes, with major implications for the conservation management of Siamese crocodiles, and provide a blueprint for the rescue effort of other "terminally ill" populations of critically endangered species.

An investigation of targeted inhibition of transcription factor activity with pyrrole imidazole polyamide (PA) in chronic myeloid leukemia (CML) blast crisis cells.

Tyrosine kinase inhibitor (TKI) therapy is the standard treatment for chronic phase (CP)-chronic myeloid leukemia (CML), yet patients in blast crisis (BC) phase of CML are unlikely to respond to TKI therapy. The transcription factor E2F1 is a down-stream target of the tyrosine kinase BCR-ABL1 and is up-regulated in TKI-resistant leukemia stem cells (LSC). Pyrrole imidazole polyamides (PA) are minor groove binders which can be programmed to target DNA sequences in a gene-selective manner. This manuscript describes such an approach with a PA designed to down-regulate E2F1 controlled gene expression by targeting a DNA sequence within 100 base pairs (bp) upstream of the E2F1 consensus sequence. Human BC-CML KCL22 cells were assessed after treatment with PA, TKI or their combination. Our PA inhibited BC-CML cell expansion based on cell density analysis compared to an untreated control after a 48-hour time-course of PA treatment. However, no evidence of cell cycle arrest was observed among BC-CML cells treated with PA, with respect to their no drug control counterparts. Thus, this work demonstrates that PAs are effective in inhibiting E2F1 TF activity which results in a temporal reduction in BC-CML cell number. We envisage that PAs could be used in the future to map genes under E2F1 control in CML LSCs.

Metabolic biomarkers of response to the AKT inhibitor MK-2206 in pre-clinical models of human colorectal and prostate carcinoma.

BACKGROUND: AKT is commonly overexpressed in tumours and plays an important role in the metabolic reprogramming of cancer. We have used magnetic resonance spectroscopy (MRS) to assess whether inhibition of AKT signalling would result in metabolic changes that could potentially be used as biomarkers to monitor response to AKT inhibition. METHODS: Cellular and metabolic effects of the allosteric AKT inhibitor MK-2206 were investigated in HT29 colon and PC3 prostate cancer cells and xenografts using flow cytometry, immunoblotting, immunohistology and MRS. RESULTS: In vitro treatment with MK-2206 inhibited AKT signalling and resulted in time-dependent alterations in glucose, glutamine and phospholipid metabolism. In vivo, MK-2206 resulted in inhibition of AKT signalling and tumour growth compared with vehicle-treated controls. In vivo MRS analysis of HT29 subcutaneous xenografts showed similar metabolic changes to those seen in vitro including decreases in the tCho/water ratio, tumour bioenergetic metabolites and changes in glutamine and glutathione metabolism. Similar phosphocholine changes compared to in vitro were confirmed in the clinically relevant orthotopic PC3 model. CONCLUSION: This MRS study suggests that choline metabolites detected in response to AKT inhibition are time and microenvironment-dependent, and may have potential as non-invasive biomarkers for monitoring response to AKT inhibitors in selected cancer types.

Local immune response to larvae of Rhipicephalus microplus in Santa Gertrudis cattle.

This study investigated the local immune response at larval attachment sites in Santa Gertrudis cattle with low and high levels of tick resistance. Skin samples with tick larvae attached were collected from Santa Gertrudis cattle at the end of a period of 25 weekly infestations, when the animals manifested highly divergent tick-resistant phenotypes. There was a tendency for more CD3+ , CD4+ , CD8+ , CD25+ , γδ T cells and neutrophils to concentrate at larval tick attachment site in susceptible cattle than in resistant cattle but the differences were significant only for γδ T cells and CD4+ cells. Most of the cattle developed intra-epidermal vesicles at the larval attachment site but the predominant cell within or around the vesicles was the neutrophil in susceptible animals and eosinophil in the resistant animals. The monoclonal antibodies (mAbs) specific for CD45 and CD45 RO antigens reacted with skin leucocytes from a higher number of susceptible cattle than resistant cattle. Our data suggest that some of the cellular responses mounted at larval attachment site are not involved in tick protection. The mAbs specific for CD45 and CD45 RO directly, or a test for CD45 genotype might be developed as markers of tick susceptibility or resistance.

First characterization of a biphasic, switch-like DNA amplification.

We report the first DNA amplification chemistry with switch-like characteristics: the chemistry is biphasic, with an expected initial phase followed by an unprecedented high gain burst of product oligonucleotide in a second phase. The first and second phases are separated by a temporary plateau, with the second phase producing 10 to 100 times more product than the first. The reaction is initiated when an oligonucleotide binds and opens a palindromic looped DNA template with two binding domains. Upon loop opening, the oligonucleotide trigger is rapidly amplified through cyclic extension and nicking of the bound trigger. Loop opening and DNA association drive the amplification reaction, such that reaction acceleration in the second phase is correlated with DNA association thermodynamics. Without a palindromic sequence, the chemistry resembles the exponential amplification reaction (EXPAR). EXPAR terminates at the initial plateau, revealing a previously unknown phenomenon that causes early reaction cessation in this popular oligonucleotide amplification reaction. Here we present two distinct types of this biphasic reaction chemistry and propose dominant reaction pathways for each type based on thermodynamic arguments. These reactions create an endogenous switch-like output that reacts to approximately 1 pM oligonucleotide trigger. The chemistry is isothermal and can be adapted to respond to a broad range of input target molecules such as proteins, genomic bacterial DNA, viral DNA, and microRNA. This rapid DNA amplification reaction could potentially impact a variety of disciplines such as synthetic biology, biosensors, DNA computing, and clinical diagnostics.