Rapid ratiometric determination of hemoglobin concentration using UV-VIS diffuse reflectance at isosbestic wavelengths.

We developed a ratiometric method capable of estimating total hemoglobin concentration from optically measured diffuse reflectance spectra. The three isosbestic wavelength ratio pairs that best correlated to total hemoglobin concentration independent of saturation and scattering were 545/390, 452/390, and 529/390 nm. These wavelength pairs were selected using forward Monte Carlo simulations which were used to extract hemoglobin concentration from experimental phantom measurements. Linear regression coefficients from the simulated data were directly applied to the phantom data, by calibrating for instrument throughput using a single phantom. Phantoms with variable scattering and hemoglobin saturation were tested with two different instruments, and the average percent errors between the expected and ratiometrically-extracted hemoglobin concentration were as low as 6.3%. A correlation of r = 0.88 between hemoglobin concentration extracted using the 529/390 nm isosbestic ratio and a scalable inverse Monte Carlo model was achieved for in vivo dysplastic cervical measurements (hemoglobin concentrations have been shown to be diagnostic for the detection of cervical pre-cancer by our group). These results indicate that use of such a simple ratiometric method has the potential to be used in clinical applications where tissue hemoglobin concentrations need to be rapidly quantified in vivo.

Enhancing imaging systems using transformation optics.

We apply the transformation optical technique to modify or improve conventional refractive and gradient index optical imaging devices. In particular, when it is known that a detector will terminate the paths of rays over some surface, more freedom is available in the transformation approach, since the wave behavior over a large portion of the domain becomes unimportant. For the analyzed configurations, quasi-conformal and conformal coordinate transformations can be used, leading to simplified constitutive parameter distributions that, in some cases, can be realized with isotropic index; index-only media can be low-loss and have broad bandwidth. We apply a coordinate transformation to flatten a Maxwell fish-eye lens, forming a near-perfect relay lens; and also flatten the focal surface associated with a conventional refractive lens, such that the system exhibits an ultra-wide field-of-view with reduced aberration.

Transformation optics with photonic band gap media.

We introduce a class of optical media based on adiabatically modulated, dielectric-only, and potentially extremely low-loss, photonic crystals (PC). The media we describe represent a generalization of the eikonal limit of transformation optics (TO). The basis of the concept is the possibility to fit some equal frequency surfaces of certain PCs with elliptic surfaces, allowing them to mimic the dispersion relation of light in anisotropic effective media. PC cloaks and other TO devices operating at visible wavelengths can be constructed from optically transparent substances such as glasses, whose attenuation coefficient can be as small as 10 dB/km, suggesting the TO design methodology can be applied to the development of optical devices not limited by the losses inherent to metal-based, passive metamaterials.

Magnetic resonance water proton relaxation in protein solutions and tissue: T(1rho) dispersion characterization.

BACKGROUND: Image contrast in clinical MRI is often determined by differences in tissue water proton relaxation behavior. However, many aspects of water proton relaxation in complex biological media, such as protein solutions and tissue are not well understood, perhaps due to the limited empirical data. PRINCIPAL FINDINGS: Water proton T(1), T(2), and T(1rho) of protein solutions and tissue were measured systematically under multiple conditions. Crosslinking or aggregation of protein decreased T(2) and T(1rho), but did not change high-field T(1). T(1rho) dispersion profiles were similar for crosslinked protein solutions, myocardial tissue, and cartilage, and exhibited power law behavior with T(1rho)(0) values that closely approximated T(2). The T(1rho) dispersion of mobile protein solutions was flat above 5 kHz, but showed a steep curve below 5 kHz that was sensitive to changes in pH. The T(1rho) dispersion of crosslinked BSA and cartilage in DMSO solvent closely resembled that of water solvent above 5 kHz but showed decreased dispersion below 5 kHz. CONCLUSIONS: Proton exchange is a minor pathway for tissue T(1) and T(1rho) relaxation above 5 kHz. Potential models for relaxation are discussed, however the same molecular mechanism appears to be responsible across 5 decades of frequencies from T(1rho) to T(1).

Utility of telomerase-pot1 fusion protein in vascular tissue engineering.

While advances in regenerative medicine and vascular tissue engineering have been substantial in recent years, important stumbling blocks remain. In particular, the limited life span of differentiated cells that are harvested from elderly human donors is an important limitation in many areas of regenerative medicine. Recently, a mutant of the human telomerase reverse transcriptase enzyme (TERT) was described, which is highly processive and elongates telomeres more rapidly than conventional telomerase. This mutant, called pot1-TERT, is a chimeric fusion between the DNA binding protein pot1 and TERT. Because pot1-TERT is highly processive, it is possible that transient delivery of this transgene to cells that are utilized in regenerative medicine applications may elongate telomeres and extend cellular life span while avoiding risks that are associated with retroviral or lentiviral vectors. In the present study, adenoviral delivery of pot1-TERT resulted in transient reconstitution of telomerase activity in human smooth muscle cells, as demonstrated by telomeric repeat amplification protocol (TRAP). In addition, human engineered vessels that were cultured using pot1-TERT-expressing cells had greater collagen content and somewhat better performance in vivo than control grafts. Hence, transient delivery of pot1-TERT to elderly human cells may be useful for increasing cellular life span and improving the functional characteristics of resultant tissue-engineered constructs.

Towards a field-compatible optical spectroscopic device for cervical cancer screening in resource-limited settings: effects of calibration and pressure.

Quantitative optical spectroscopy has the potential to provide an effective low cost, and portable solution for cervical pre-cancer screening in resource-limited communities. However, clinical studies to validate the use of this technology in resource-limited settings require low power consumption and good quality control that is minimally influenced by the operator or variable environmental conditions in the field. The goal of this study was to evaluate the effects of two sources of potential error: calibration and pressure on the extraction of absorption and scattering properties of normal cervical tissues in a resource-limited setting in Leogane, Haiti. Our results show that self-calibrated measurements improved scattering measurements through real-time correction of system drift, in addition to minimizing the time required for post-calibration. Variations in pressure (tested without the potential confounding effects of calibration error) caused local changes in vasculature and scatterer density that significantly impacted the tissue absorption and scattering properties Future spectroscopic systems intended for clinical use, particularly where operator training is not viable and environmental conditions unpredictable, should incorporate a real-time self-calibration channel and collect diffuse reflectance spectra at a consistent pressure to maximize data integrity.

Instrument independent diffuse reflectance spectroscopy.

Diffuse reflectance spectroscopy with a fiber optic probe is a powerful tool for quantitative tissue characterization and disease diagnosis. Significant systematic errors can arise in the measured reflectance spectra and thus in the derived tissue physiological and morphological parameters due to real-time instrument fluctuations. We demonstrate a novel fiber optic probe with real-time, self-calibration capability that can be used for UV-visible diffuse reflectance spectroscopy in biological tissue in clinical settings. The probe is tested in a number of synthetic liquid phantoms over a wide range of tissue optical properties for significant variations in source intensity fluctuations caused by instrument warm up and day-to-day drift. While the accuracy for extraction of absorber concentrations is comparable to that achieved with the traditional calibration (with a reflectance standard), the accuracy for extraction of reduced scattering coefficients is significantly improved with the self-calibration probe compared to traditional calibration. This technology could be used to achieve instrument-independent diffuse reflectance spectroscopy in vivo and obviate the need for instrument warm up and post∕premeasurement calibration, thus saving up to an hour of precious clinical time.

Portable, Fiber-Based, Diffuse Reflection Spectroscopy (DRS) Systems for Estimating Tissue Optical Properties.

Steady-state diffuse reflection spectroscopy is a well-studied optical technique that can provide a noninvasive and quantitative method for characterizing the absorption and scattering properties of biological tissues. Here, we compare three fiber-based diffuse reflection spectroscopy systems that were assembled to create a light-weight, portable, and robust optical spectrometer that could be easily translated for repeated and reliable use in mobile settings. The three systems were built using a broadband light source and a compact, commercially available spectrograph. We tested two different light sources and two spectrographs (manufactured by two different vendors). The assembled systems were characterized by their signal-to-noise ratios, the source-intensity drifts, and detector linearity. We quantified the performance of these instruments in extracting optical properties from diffuse reflectance spectra in tissue-mimicking liquid phantoms with well-controlled optical absorption and scattering coefficients. We show that all assembled systems were able to extract the optical absorption and scattering properties with errors less than 10%, while providing greater than ten-fold decrease in footprint and cost (relative to a previously well-characterized and widely used commercial system). Finally, we demonstrate the use of these small systems to measure optical biomarkers in vivo in a small-animal model cancer therapy study. We show that optical measurements from the simple portable system provide estimates of tumor oxygen saturation similar to those detected using the commercial system in murine tumor models of head and neck cancer.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48-90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48-65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm(2), respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.

Developmental and tissue-specific expression of NITRs.

Novel immune-type receptors (NITRs) are encoded by large multi-gene families and share structural and signaling similarities to mammalian natural killer receptors (NKRs). NITRs have been identified in multiple bony fish species, including zebrafish, and may be restricted to this large taxonomic group. Thirty-nine NITR genes that can be classified into 14 families are encoded on zebrafish chromosomes 7 and 14. Herein, we demonstrate the expression of multiple NITR genes in the zebrafish ovary and during embryogenesis. All 14 families of zebrafish NITRs are expressed in hematopoietic kidney, spleen and intestine as are immunoglobulin and T cell antigen receptors. Furthermore, all 14 families of NITRs are shown to be expressed in the lymphocyte lineage, but not in the myeloid lineage, consistent with the hypothesis that NITRs function as NKRs. Sequence analyses of NITR amplicons identify known alleles and reveal additional alleles within the nitr1, nitr2, nitr3, and nitr5 families, reflecting the recent evolution of this gene family.

The controlled delivery of hydrogen sulfide for the preservation of heart tissue

Gemstone Team Organ Storage and Hibernation

Radioactive in situ hybridization for detecting diverse gene expression patterns in tissue.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)(1) that has been working successfully in our lab for many years, especially for adult vertebrate brains(2-5). The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts(6,7). Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches(8,9), in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity.

Immune responses must be well restrained in a steady state to avoid excessive inflammation. However, such restraints are quickly removed to exert antimicrobial responses. Here we report a role of autophagy in an early host antifungal response by enhancing NFκB activity through A20 sequestration. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80(hi) macrophages in the spleen, peritoneum and kidney. We show that p62, an autophagic adaptor protein, captures A20 to sequester it in the autophagosome. This allows the macrophages to release chemokines to recruit neutrophils. Indeed, mice lacking autophagy in myeloid cells show higher susceptibility to Candida albicans infection due to impairment in neutrophil recruitment. Thus, at least in the specific aforementioned tissues, autophagy appears to break A20-dependent suppression in F4/80(hi) macrophages, which express abundant A20 and contribute to the initiation of efficient innate immune responses.

Bioluminescence imaging of glucose in tissue surrounding polyurethane and glucose sensor implants.

BACKGROUND: The bioluminescence technique was used to quantify the local glucose concentration in the tissue surrounding subcutaneously implanted polyurethane material and surrounding glucose sensors. In addition, some implants were coated with a single layer of adipose-derived stromal cells (ASCs) because these cells improve the wound-healing response around biomaterials. METHODS: Control and ASC-coated implants were implanted subcutaneously in rats for 1 or 8 weeks (polyurethane) or for 1 week only (glucose sensors). Tissue biopsies adjacent to the implant were immediately frozen at the time of explant. Cryosections were assayed for glucose concentration profile using the bioluminescence technique. RESULTS: For the polyurethane samples, no significant differences in glucose concentration within 100 μm of the implant surface were found between bare and ASC-coated implants at 1 or 8 weeks. A glucose concentration gradient was demonstrated around the glucose sensors. For all sensors, the minimum glucose concentration of approximately 4 mM was found at the implant surface and increased with distance from the sensor surface until the glucose concentration peaked at approximately 7 mM at 100 μm. Then the glucose concentration decreased to 5.5-6.5 mM more than 100 μmm from the surface. CONCLUSIONS: The ASC attachment to polyurethane and to glucose sensors did not change the glucose profiles in the tissue surrounding the implants. Although most glucose sensors incorporate a diffusion barrier to reduce the gradient of glucose and oxygen in the tissue, it is typically assumed that there is no steep glucose gradient around the sensors. However, a glucose gradient was observed around the sensors. A more complete understanding of glucose transport and concentration gradients around sensors is critical.