Machine learning enabled multiplex detection of periodontal pathogens by surface-enhanced Raman spectroscopy.

Periodontitis is a chronic inflammation of the periodontium caused by a persistent bacterial infection, resulting in destruction of the supporting structures of teeth. Analysis of microbial composition in saliva can inform periodontal status. Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Streptococcus mutans (Sm) are among reported periodontal pathogens, and were used as model systems in this study. Our atomic force microscopic (AFM) study revealed that these pathogens are biological nanorods with dimensions of 0.6-1.1 µm in length and 500-700 nm in width. Current bacterial detection methods often involve complex preparation steps and require labeled reporting motifs. Employing surface-enhanced Raman spectroscopy (SERS), we revealed cell-type specific Raman signatures of these pathogens for label-free detection. It overcame the complexity associated with spectral overlaps among different bacterial species, relying on high signal-to-noise ratio (SNR) spectra carefully collected from pure species samples. To enable simple, rapid, and multiplexed detection, we harnessed advanced machine learning techniques to establish predictive models based on a large set of raw spectra of each bacterial species and their mixtures. Using these models, given a raw spectrum collected from a bacterial suspension, simultaneous identification of all three species in the test sample was achieved at 95.6 % accuracy. This sensing modality can be applied to multiplex detection of a broader range and a larger set of periodontal pathogens, paving the way for hassle-free detection of oral bacteria in saliva with little to no sample preparation.

Survivin-Sodium Iodide Symporter Reporter as a Non-Invasive Diagnostic Marker to Differentiate Uterine Leiomyosarcoma from Leiomyoma.

Leiomyosarcoma (LMS) has been challenging to diagnose because of limitations in clinical and radiographic predictors, as well as the lack of reliable serum or urinary biomarkers. Most uterine masses consist of benign leiomyoma (LM). However, it is currently a significant challenge in gynecology practice to differentiate LMS from LM. This inability poses grave consequences for patients, leading to a high number of unnecessary hysterectomies, infertility, and other major morbidities and possible mortalities. This study aimed to evaluate the use of Survivin-Sodium iodide symporter (Ad-Sur-NIS) as a reporter gene biomarker to differentiate malignant LMS from benign LM by using an F18-NaBF4 PET/CT scan. The PET/CT scan images showed a significantly increased radiotracer uptake and a decreased radiotracer decay attributable to the higher abundance of Ad-Sur-NIS in the LMS tumors compared to LM (p < 0.05). An excellent safety profile was observed, with no pathological or metabolic differences detected in Ad-Sur-NIS-treated animal versus the vehicle control. Ad-Sur-NIS as a PET scan reporter is a promising imaging biomarker that can differentiate uterine LMS from LM using F18-NaBF4 as a radiotracer. As a new diagnostic method, the F18 NaBF4 PET/CT scan can provide a much-needed tool in clinical practices to effectively triage women with suspicious uterine masses and avoid unnecessary invasive interventions.

Role of photobleaching process of indocyanine green for killing neuroblastoma cells.

Indocyanine green (ICG) is an FDA-approved near infrared (NIR) imaging agent for diagnosis and imaging guided surgery. It also exhibits phototoxicity under high-dose NIR irradiation, expanding its application as a photo-therapeutic agent. Since ICG's efficiency as a type II photosensitizer has been controversial due to its low triplet state yield, other mechanisms have been explored. While claims of toxic decomposition products, accompanied by irreversible ICG photobleaching, were proposed as the main mechanism, evidences from systemic studies are lacking. In this work, we aimed to unravel the factors affecting ICG photobleaching and the associated photo-killing effect on neuroblastoma, one of the most common pediatric tumors but often escapes therapy. Specifically, we examined how albumin-induced ICG stabilization affects the ICG photobleaching process, and the effect of photobleached ICG on cell proliferation and viability of neuroblastoma cells. It was found that ICG photobleaching was significant only under aerobic conditions and was more efficient in solutions with higher concentration ICG monomers, which were stabilized from aggregates by the presence of BSA while increasing photobleaching and associated oxygen consumption. Photobleached ICG inhibited cell proliferation, indicating another effect of tumor treatment by ICG. Taken together, while enhanced photobleaching by BSA-bound ICG monomers may reduce the photodynamic effect targeting cellular components, the photoproducts directly contribute to tumor growth inhibition and assist in a secondary mechanism to stop tumor growth.

Robotic-Assisted Roux-en-Y Gastric Bypass: Learning Curve Assessment Using Cumulative Sum and Literature Review.

Purpose: Robotic-assisted Roux-en-Y gastric bypass (RARYGB) is a procedure that is used with increasing frequency in the United States. Among other bariatric procedures, RARYGB is a good model for the robotic platform because it allows hand-sewn suturing and energy devices application. The aim of this study was to conduct a literature review of robotic approach in RARYGB, its learning curve using the cumulative sum (CUSUM) method, and our experience as Center of Excellence recognized by the American Society for Metabolic and Bariatric Surgery (ASMBS). Methods: A total of 67 patients were included. Results revealed that the learning curve was achieved after case 11. Eighteen studies were included in the pooled analysis. Results: An increase in the operative time was noted at case 46, in which a second phase was identified. A significant difference between these two phases was found only related to previous bariatric surgery. The outcomes of this series were comparable with the ones available in the literature. Conclusions: The robotic platform is increasing its role in complex procedures such as RARYGB. The hand-sewn technique may offer important advantages in terms of shorter learning curve, reduced conversion rate, and lower leakage rate.

Near-Infrared Indocyanine Green-Enhanced Fluorescence and Evaluation of the Bowel Microperfusion During Robotic Colorectal Surgery: a Retrospective Original Paper.

BACKGROUND: Leakage of the anastomosis after colorectal surgery is a severe complication, and one of the most important causes is poor vascular supply. However, a microvascular deficit is often not detectable during surgery under white light. Near-infrared indocyanine green (ICG)-enhanced fluorescence may be useful for assessing microvascular deficits and conceivably preventing anastomotic leakage. OBJECTIVES: This paper presents a preliminary retrospective case series on robotic colorectal surgery. The aim is to evaluate the feasibility, safety and role of near-infrared ICG-enhanced ?uorescence for the intraoperative assessment of peri-anastomotic tissue vascular perfusion. MATERIALS AND METHODS: From among more than 164 robotic colorectal cases performed, we retrospectively analyzed 28 that were all performed by the same surgeon (PCG) using near-infrared ICG-enhanced fluorescence technology: 16 left colectomies (57.1%), 8 rectal resections (28.6%), 3 right colectomies (10.8%) and 1 pancolectomy (3.6%). RESULTS: The rates of conversion, intraoperative complications, dye allergic reaction and mortality were all 0%. In two cases (7.1%)-1 left and 1 right colectomy-the level of the anastomosis was changed intraoperatively after ICG showed ischemic tissues. Despite the application of ICG, one anastomotic leak (after left colectomy for a chronic recurrent sigmoid diverticulitis with pericolic abscess) was observed. CONCLUSIONS: ICG technology may help to determine when to intraoperatively change the anastomotic level to a safer location. In our case series, ICG results led to a change in the level of the anastomosis in 7.1% of the cases. Despite the use of ICG, we observed one leak. This may have been related to vascularization-independent causes (e.g., infection in this case) or may reflect a need for better standardization of this ICG technology. In particular, we need a way to objectively assess the ICG signal and the related risk of leakage. More randomized, prospective, well-powered trials are needed to unveil the full potential of this innovative surgical technology.

Combined application of Indocyanine green (ICG) and laser lead to targeted tumor cell destruction.

PURPOSE: Precise excision of neuroblastoma is challenging, especially when tumors adhere to vital structures. Indocyanine green (ICG), an FDA-approved dye with absorption peaking at 800 nm, can absorb the near IR laser energy and release heat in the dyed tissue. We hypothesize that by injecting ICG at tumor sites followed by precise laser application, tumor cell death can be selectively targeted. METHODS: Orthotopic neuroblastoma tumors were created in the adrenal gland of immunocompromised mice. Tumor, liver, kidney, and muscle tissues were chosen for ICG injection. Intervention variables included presence of tumor capsule, continuous vs. pulsed laser treatment and total energy delivered. Control groups included laser or ICG only. Tissues were stained with hematoxylin/eosin. RESULTS: Continuous wave laser generated excessive heat, causing damage in all tissues. When using pulsed laser treatment, liver, kidney, muscle, and intact tumor tissues showed no cell death when treated with laser alone or laser plus ICG. Tumor tissue with the capsule removed, however, showed cell death on histology. CONCLUSIONS: Pulsed laser treatment combined with ICG causes targeted tumor cell death in neuroblastoma tumor without capsule. No cell death was observed when tumor capsule was present, when only laser was used, or when applied over non-tumor tissues.

Near-Infrared Indocyanine Green-Enhanced Fluorescence and Minimally Invasive Colorectal Surgery: Review of the Literature.

BACKGROUND: Leakage of the anastomosis after colonic/rectal surgery is a serious complication. One of the most important causes of anastomotic leakage is impaired vascularization. A microvascular tissue deficit is very often not intraoperatively de visu detectable under white light. Near-infrared indocyanine green (ICG)-enhanced fluorescence is a cutting-edge technology that may be useful for detecting microvascular impairment and potentially preventing anastomotic leakage. AIM: The aim of this narrative review was to evaluate the feasibility and the usefulness of intraoperative assessment of vascular anastomotic perfusion in colorectal surgery using an indocyanine green (ICG) fluorescent tracer. MATERIAL AND METHODS: A PubMed/MedLine, Embase, and Scopus narrative literature review was performed, in which "colorectal surgery" and "indocyanine green" were used as key words. The inclusion criteria were 1) manuscripts written in English; 2) full text is available; 3) topic related to the use of ICG fluorescence for the assessment of tissue perfusion during laparoscopic or robotic colorectal surgery; and 4) sample: adult patients, benign or malignant disease. Exclusion criteria included 1) case reports; 2) topic not related to the use of ICG fluorescence for the evaluation of tissue perfusion during laparoscopic or robotic colorectal surgery; 3) manuscripts that focused solely on other applications of ICG technology; and 4) any study type not showing original data. Results and Critical Discussion: The intraoperative visual assessment of tissue viability under white light may lead to an underestimation of microvascular blood flow impairment. ICG can be safely used in cases of minimally invasive colonic surgery and also low anterior resections. This technology may be useful when deciding whether to intraoperatively change a previously planned resection/anastomotic level, which could decrease theoretically the occurrence of anastomotic leakage. CONCLUSIONS: Near-infrared ICG technology is a very useful approach. Multiple preliminary studies suggest that this technique may be used to predict anastomotic leakage. However, evaluation of the ICG signal is still too subjective. Some reliable scoring/grading parameters related to the ICG signal need to be defined. Additionally, more prospective, randomized, and adequately powered studies are required to completely reveal the true potential of this surgical technological innovation.

Indocyanine Green (Icg)-Enhanced Fluorescence for Intraoperative Assessment of Bowel Microperfusion During Laparoscopic and Robotic Colorectal Surgery: The Quest for Evidence-Based Results.

Anastomotic leakage is a severe complication after colonic/rectal surgery. One of the most important causes of anastomotic leakage is poor vascular supply. However, microvascular impairment at the anastomotic site is very often not detected intraoperatively by observation under white light. Indocyanine green (ICG)-enhanced fluorescence is a technology that may be useful for detecting microvascular alterations and potentially preventing anastomotic leakage. The aim of this Editorial-Minireview is to briefly and critically assess the literature evidence regarding the feasibility of using an ICG ?uorescent tracer for detecting microvascular changes in the perianastomotic tissue and its potential role in preventing anastomotic leakage. We focused on minimally invasive (robotic and laparoscopic) colorectal surgery. Intraoperative ICG angiography and the quantification of ICG kinetics can be used to intraoperatively reveal the tissue-perfusion status during colorectal surgery. This may be useful for intraoperatively changing a previously planned resection/anastomotic level, and conceivably decreasing the degree of anastomotic leakage. At this stage, even though ICG technology appears to be very promising and some preliminary clinical studies have suggested that certain ICG pharmacokinetic parameters may be used to predict leakage, more reliable scoring and grading tools are needed. Furthermore, in minimally invasive colorectal surgery, more randomized prospective well-powered trials are needed to properly standardize this surgical technology.

New generation of electrochemical immunoassay based on polymeric nanoparticles for early detection of breast cancer.

Screening and early diagnosis are the key factors for the reduction of mortality rate and treatment cost of cancer. Therefore, sensitive and selective methods that can reveal the low abundance of cancer biomarkers in a biological sample are always desired. Here, we report the development of a novel electrochemical biosensor for early detection of breast cancer by using bioconjugated self-assembled pH-responsive polymeric micelles. The micelles were loaded with ferrocene molecules as "tracers" to specifically target cell surface-associated epithelial mucin (MUC1), a biomarker for breast and other solid carcinoma. The synthesis of target-specific, ferrocene-loaded polymeric micelles was confirmed, and the resulting sensor was capable of detecting the presence of MUC1 in a sample containing about 10 cells/mL. Such a high sensitivity was achieved by maximizing the loading capacity of ferrocene inside the polymeric micelles. Every single event of binding between the antibody and antigen was represented by the signal of hundreds of thousands of ferrocene molecules that were released from the polymeric micelles. This resulted in a significant increase in the intensity of the ferrocene signal detected by cyclic voltammetry.

Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

Target-specific copper hybrid T7 phage particles.

Target-specific nanoparticles have attracted significant attention recently, and have greatly impacted life and physical sciences as new agents for imaging, diagnosis, and therapy, as well as building blocks for the assembly of novel complex materials. While most of these particles are synthesized by chemical conjugation of an affinity reagent to polymer or inorganic nanoparticles, we are promoting the use of phage particles as a carrier to host organic or inorganic functional components, as well as to display the affinity reagent on the phage surface, taking advantage of the fact that some phages host well-established vectors for protein expression. An affinity reagent can be structured in a desired geometry on the surface of phage particles, and more importantly, the number of the affinity reagent molecules per phage particle can be precisely controlled. We previously have reported the use of the T7 phage capsid as a template for synthesizing target-specific metal nanoparticles. In this study herein, we reported the synthesis of nanoparticles using an intact T7 phage as a scaffold from which to extend 415 copies of a peptide that contains a hexahistidine (6His) motif for capture of copper ions and staging the conversion of copper ions to copper metal, and a cyclic Arginine-Glycine-Aspartic Acid (RGD4C) motif for targeting integrin and cancer cells. We demonstrated that the recombinant phage could load copper ions under low bulk copper concentrations without interfering with its target specificity. Further reduction of copper ions to copper metal rendered a very stable copper hybrid T7 phage, which prevents the detachment of copper from phage particles and maintains the phage structural integrity even under harsh conditions. Cancer cells (MCF-7) can selectively uptake copper hybrid T7 phage particles through ligand-mediated transmembrane transportation, whereas normal control cells (MCF-12F) uptake 1000-fold less. We further demonstrated that copper hybrid T7 phage could be endocytosed by cancer cells in culture.

Traceless cross-linker for photocleavable bioconjugation.

Photoresponsive bioconjugation empowers the development of novel methods for drug discovery, disease diagnosis, and high-throughput screening, among others. In this paper, we report on the characteristics of a traceless photocleavable cross-linker, di-6-(3-succinimidyl carbonyloxymethyl-4-nitro-phenoxy)-hexanoic acid disulfide diethanol ester (SCNE). The traceless feature and the biocompatibility of this photocleavable cross-linking reagent were corroborated. Consequently, we demonstrated its application in reversible phage particle immobilization that could provide a platform for direct single-phage screening. We also applied it in protein-photoprinting, where SCNE acts as a "photo-eraser" to remove the cross-linked protein molecules at a desired region in a simple, clean, and light-controllable fashion. We further demonstrated the two-tier atomic force microscopic (AFM) method that uses SCNE to carry out two subsequent AFM tasks in situ. The approach allows guided protein delivery and subsequent high-resolution imaging at the same local area, thus opening up the possibility of monitoring protein functions in live cells. The results imply that SCNE is a versatile cross-linker that can be used for a wide range of applications where photocleavage ensures clean and remote-controllable release of biological molecules from a substrate.

Cell growth on different types of ultrananocrystalline diamond thin films.

Unique functional materials provide a platform as scaffolds for cell/tissue regeneration. Investigation of cell-materials' chemical and biological interactions will enable the application of more functional materials in the area of bioengineering, which provides a pathway to the novel treatment for patients who suffer from tissue/organ damage and face the limitation of donation sources. Many studies have been made into tissue/organ regeneration. Development of new substrate materials as platforms for cell/tissue regeneration is a key research area. Studies discussed in this paper focus on the investigation of novel ultrananocrystalline diamond (UNCD) films as substrate/scaffold materials for developmental biology. Specially designed quartz dishes have been coated with different types of UNCD films and cells were subsequently seeded on those films. Results showed the cells' growth on UNCD-coated culture dishes are similar to cell culture dishes with little retardation, indicating that UNCD films have no or little inhibition on cell proliferation and are potentially appealing as substrate/scaffold materials. The mechanisms of cell adhesion on UNCD surfaces are proposed based on the experimental results. The comparisons of cell cultures on diamond-powder-seeded culture dishes and on UNCD-coated dishes with matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy (MALDI-TOF MS) and X-ray photoelectron spectroscopy (XPS) analyses provided valuable data to support the mechanisms proposed to explain the adhesion and proliferation of cells on the surface of the UNCD platform.

Trackable and Targeted Phage as Positron Emission Tomography (PET) Agent for Cancer Imaging.

UNLABELLED: The recent advancement of nanotechnology has provided unprecedented opportunities for the development of nanoparticle enabled technologies for detecting and treating cancer. Here, we reported the construction of a PET trackable organic nanoplatform based on phage particle for targeted tumor imaging. METHOD: The integrin α(v)ß(3) targeted phage nanoparticle was constructed by expressing RGD peptides on its surface. The target binding affinity of this engineered phage particle was evaluated in vitro. A bifunctional chelator (BFC) 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) or 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) was then conjugated to the phage surface for (64)Cu(2+) chelation. After (64)Cu radiolabeling, microPET imaging was performed in U87MG tumor model and the receptor specificity was confirmed by blocking experiments. RESULTS: The phage-RGD demonstrated target specificity based on ELISA experiment. According to the TEM images, the morphology of the phage was unchanged after the modification with BFCs. The labeling yield was 25 ± 4% for (64)Cu-DOTA-phage-RGD and 46 ± 5% for (64)Cu-AmBaSar-phage-RGD, respectively. At 1 h time point, (64)Cu-DOTA-phage-RGD and (64)Cu-AmBaSar-phage-RGD have comparable tumor uptake (~ 8%ID/g). However, (64)Cu-AmBaSar-phage-RGD showed significantly higher tumor uptake (13.2 ± 1.5 %ID/g, P<0.05) at late time points compared with (64)Cu-DOTA-phage-RGD (10 ± 1.2 %ID/g). (64)Cu-AmBaSar-phage-RGD also demonstrated significantly lower liver uptake, which could be attributed to the stability difference between these chelators. There is no significant difference between two tracers regarding the uptake in kidney and muscle at all time points tested. In order to confirm the receptor specificity, blocking experiment was performed. In the RGD blocking experiment, the cold RGD peptide was injected 2 min before the administration of (64)Cu-AmBaSar-phage-RGD. Tumor uptake was partially blocked at 1 h time point. Phage-RGD particle was also used as the competitive ligand. In this case, the tumor uptake was significantly reduced and the value was kept at low level consistently. CONCLUSION: In this report, we constructed a PET trackable nanoplatform based on phage particle and demonstrated the imaging capability of these targeted agents. We also demonstrated that the choice of chelator could have significant impact on imaging results of nano-agents. The method established in this research may be applicable to other receptor/ligand systems for theranostic agent construction, which could have an immediate and profound impact on the field of imaging/therapy and lay the foundation for the construction of next generation cancer specific theranostic agents.

Loss of pluripotency in human embryonic stem cells directly correlates with an increase in nuclear zinc.

The pluripotency of human embryonic stem cells (hESCs) is important to investigations of early development and to cell replacement therapy, but the mechanism behind pluripotency is incompletely understood. Zinc has been shown to play a key role in differentiation of non-pluripotent cell types, but here its role in hESCs is directly examined. By mapping the distribution of metals in hESCs at high resolution by x-ray fluorescence microprobe (XFM) and by analyzing subcellular metal content, we have found evidence that loss of pluripotency is directly correlated with an increase in nuclear zinc. Zinc elevation not only redefines our understanding of the mechanisms that support pluripotency, but also may act as a biomarker and an intervention point for stem cell differentiation.

Random mitotic activities across human embryonic stem cell colonies.

A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

High Fidelity Nano-Hole Enhanced Raman Spectroscopy.

Surface Enhanced Raman Spectroscopy (SERS) is a sensitive technique that can even detect single molecules. However, in many SERS applications, the strongly inhomogeneous distribution of intense local fields makes it very difficult for a quantitive assessment of the fidelity, or reproducibility of the signal, which limits the application of SERS. Herein we report the development of exceptionally high fidelity Hole-Enhanced Raman Spectroscopy (HERS) from ordered, two-dimensional hexagonal nanohole arrays. We take the fidelity f to be a measure of the percent deviation of the Raman peaks from measurement to measurement. Overall, area averaged fidelities for 12 gold array samples ranged from f ~ 2% - 15% for HERS using aqueous R6G molecules. Furthermore, intensity modulations of the enhanced Raman spectra were measured for the first time as a function of polarization angle. The best of these measurements, which focus on static laser spots on the sample, could be consistent with even higher fidelities than the area-averaged results. Nanohole arrays in silver provided supporting polarization measurements and a more complete enhanced Raman fingerprint for phenylalanine molecules. We also carried out finite-difference time-domain calculations to assist in the interpretation of the experiments, identifying the polarization dependence as possibly arising from hole-hole interactions. Our results represent a step towards making quantitative and reproducible enhanced Raman measurements possible and also open new avenues for a large scale source of highly uniform hot spots.

Profiling TRA-1-81 antigen distribution on a human embryonic stem cell.

Human embryonic stem (hES) cells hold great promise in regenerative medicine. Although hES cells have unlimited self-renewal potential, they tend to differentiate spontaneously in culture. TRA-1-81 is a biomarker of undifferentiated hES cells. Quantitative characterization of TRA-1-81 expression level in a single cell helps capture the "turn-on" signal and understand the mechanism of early differentiation. Here, we report on our examination of TRA-1-81 distribution and association on a hES cell membrane using an atomic force microscope (AFM). Our results suggest that aggregated distribution of TRA-1-81 antigen is characteristic for undifferentiated hES cells. We also evaluated the TRA-1-81 expression level at approximately 17,800 epitopes and approximately 700 epitopes per cell on an undifferentiated cell and a spontaneously differentiated cell, respectively. The method in this study can be adapted in examining other surface proteins on various cell types, thus providing a general tool for investigating protein distribution and association at the single cell level.

Metabolic photofragmentation kinetics for a minimal protocell: rate-limiting factors, efficiency, and implications for evolution.

A key requirement of an autonomous self-replicating molecular machine, a protocell, is the ability to digest resources and turn them into building blocks. Thus a protocell needs a set of metabolic processes fueled by external free energy in the form of available chemical redox potential or light. We introduce and investigate a minimal photodriven metabolic system, which is based on photofragmentation of resource molecules catalyzed by genetic molecules. We represent and analyze the full metabolic set of reaction-kinetic equations and, through a set of approximations, simplify the reaction kinetics so that analytical expressions can be obtained for the building block production. The analytical approximations are compared with the full equation set and with corresponding experimental results to the extent they are available. It should be noted, however, that the proposed metabolic system has not been experimentally implemented, so this investigation is conducted to obtain a deeper understanding of its dynamics and perhaps to anticipate its limitations. We demonstrate that this type of minimal photodriven metabolic scheme is typically rate-limited by the front-end photoexcitation process, while its yield is determined by the genetic catalysis. We further predict that gene-catalyzed metabolic reactions can undergo evolutionary selection only for certain combinations of the involved reaction rates due to their intricate interactions. We finally discuss how the expected range of metabolic rates likely affects other key protocellular processes such as container growth and division as well as gene replication.

Polymeric micelle-based bioassay with femtomolar sensitivity.

Target-specific polymeric micelles loaded with fluorescence dye molecules in their hydrophobic cores were made from block copolymer of poly(caprolactones)23-b-poly(ethylene oxide)45. It was found that the micelles are stable against pH changes from pH 2 to 12 and temperature variation up to 65 degrees C. The dye molecules can be released to the solution on exposing the micelles to organic solvents or ultrasound. A rapid and highly sensitive immunoassay based on the above micelles was developed, and the assay can detect specific target proteins in the femtomolar range from complex biological samples such as serum mimics and cell lysate. For example, less than 0.15 U/ml of ovarian cancer-specific antigen 125, equivalent to 7.5 x 10(-15)M, can be reliably detected in solution. We also demonstrated that the assay can detect a cell surface biomarker, stage-specific embryonic antigen 4, from a single human embryonic stem cell.