The impact of early RPE cell junction loss on VEGF, Ang-2, and TIMP secretion in vitro.

Purpose: The retinal pigment epithelium (RPE) is an important tissue for maintaining a healthy retina. Retinal pigment epithelial cells help regulate nutrient transport to photoreceptors and are heavily pigmented to prevent light scattering. These cells also have junction proteins to form monolayers. Monolayers are key players in pathologies such as age-related macular degeneration (AMD), a leading cause of vision loss in older adults. During AMD, RPE cell detachment can occur, resulting in a loss of junctions. Losing junctions can increase the expression of pro-angiogenic vascular endothelial growth factor (VEGF). This overexpression can cause abnormal blood vessel growth or angiogenesis in the retina. Age-related macular degeneration treatments target VEGF to slow angiogenesis progression. However, other proteins, such as angiopoietin-2 (Ang-2) and the tissue inhibitor of metalloproteinase-1 (TIMP-1), may also play important roles, making them potential targets for treatment. Controlling RPE junction formation will help elucidate the relationship between RPE cell detachment and additional angiogenic factor secretion, lead to more therapeutics, and increase the efficacy of current treatments. Methods: Micropatterning was used to control the spatial arrangement of primary porcine RPE cells using polydimethylsiloxane (PDMS) stencils. Patterns were formed into PDMS stencils to mimic 10%, 25%, and 50% overall detachment of the RPE monolayer. Zonula-occludens-1 (ZO-1), Ang-2, and VEGF were visualized using immunocytochemical (ICC) staining. An enzyme-linked immunosorbent assay (ELISA) was used to quantify extracellular Ang-2, VEGF, TIMP-1, and TIMP-2 levels. A rod outer segment (OS) phagocytosis assay was performed to determine how RPE junction loss directly affects photoreceptor support. Results: The growth of primary porcine RPE cells was successfully controlled using stencils. Morphological changes and a decrease in pigmentation were observed, showing a decline in barrier and light absorption functions as degeneration increased. One day after stencil removal, junction proteins were delocalized, and angiogenic factor secretions were correlated with increased levels of detachment. Secretion levels of Ang-2 and TIMP-1 were significantly increased, whereas VEGF and TIMP-2 concentrations were not as affected by varying levels of detachment. OS phagocytosis appeared lower in RPE cells when ZO-1 was affected. Conclusions: These results suggest a correlation between loss of junctions, abnormal angiogenic protein secretion, and reduced OS phagocytosis. Furthermore, Ang-2 and TIMP-1 proteins might be beneficial targets for AMD treatments, and their roles in retinal diseases deserve further investigation.

Simultaneous isolation and label-free identification of bacteria using contactless dielectrophoresis and Raman spectroscopy.

The traditional bacterial identification method of growing colonies on agar plates can take several days to weeks to complete depending on the growth rate of the bacteria. Successfully decreasing this analysis time requires cell isolation followed by identification. One way to decrease analysis time is by combining dielectrophoresis (DEP), a common technique used for cell sorting and isolation, and Raman spectroscopy for cell identification. DEP-Raman devices have been used for bacterial analysis, however, these devices have a number of drawbacks including sample heating, cell-to-electrode proximity that limits throughput and separation efficiency, electrode fouling, or inability to address sample debris. Presented here is a contactless DEP-Raman device to simultaneously isolate and identify particles from a mixed sample while avoiding common drawbacks associated with other DEP designs. Using the device, a mixed sample of bacteria and 3 µm polystyrene spheres were isolated from each other and a Raman spectrum of the trapped bacteria was acquired, indicating the potential for cDEP-Raman devices to decrease the analysis time of bacteria.

In vivo Raman spectroscopy for biochemical monitoring of the human cervix throughout pregnancy.

BACKGROUND: The cervix must undergo significant biochemical remodeling to allow for successful parturition. This process is not fully understood, especially in instances of spontaneous preterm birth. In vivo Raman spectroscopy is an optical technique that can be used to investigate the biochemical composition of tissue longitudinally and noninvasively in human beings, and has been utilized to measure physiology and disease states in a variety of medical applications. OBJECTIVE: The purpose of this study is to measure in vivo Raman spectra of the cervix throughout pregnancy in women, and to identify biochemical markers that change with the preparation for delivery and postpartum repair. STUDY DESIGN: In all, 68 healthy pregnant women were recruited. Raman spectra were measured from the cervix of each patient monthly in the first and second trimesters, weekly in the third trimester, and at the 6-week postpartum visit. Raman spectra were measured using an in vivo Raman system with an optical fiber probe to excite the tissue with 785 nm light. A spectral model was developed to highlight spectral regions that undergo the most changes throughout pregnancy, which were subsequently used for identifying Raman peaks for further analysis. These peaks were analyzed longitudinally to determine if they underwent significant changes over the course of pregnancy (P < .05). Finally, 6 individual components that comprise key biochemical constituents of the human cervix were measured to extract their contributions in spectral changes throughout pregnancy using a linear combination method. Patient factors including body mass index and parity were included as variables in these analyses. RESULTS: Raman peaks indicative of extracellular matrix proteins (1248 and 1254 cm-1) significantly decreased (P < .05), while peaks corresponding to blood (1233 and 1563 cm-1) significantly increased (P < .0005) in a linear manner throughout pregnancy. In the postpartum cervix, significant increases in peaks corresponding to actin (1003, 1339, and 1657 cm-1) and cholesterol (1447 cm-1) were observed when compared to late gestation, while signatures from blood significantly decreased. Postpartum actin signals were significantly higher than early pregnancy, whereas extracellular matrix proteins and water signals were significantly lower than early weeks of gestation. Parity had a significant effect on blood and extracellular matrix protein signals, with nulliparous patients having significant increases in blood signals throughout pregnancy, and higher extracellular matrix protein signals in early pregnancy compared to patients with prior pregnancies. Body mass index significantly affected actin signal contribution, with low body mass index patients showing decreasing actin contribution throughout pregnancy and high body mass index patients demonstrating increasing actin signals. CONCLUSION: Raman spectroscopy was successfully used to biochemically monitor cervical remodeling in pregnant women during prenatal visits. This foundational study has demonstrated sensitivity to known biochemical dynamics that occur during cervical remodeling, and identified patient variables that have significant effects on Raman spectra throughout pregnancy. Raman spectroscopy has the potential to improve our understanding of cervical maturation, and be used as a noninvasive preterm birth risk assessment tool to reduce the incidence, morbidity, and mortality caused by preterm birth.

Physical disruption of cell-cell contact induces VEGF expression in RPE cells.

PURPOSE: To investigate the role of RPE cell-cell contact in vascular endothelial growth factor (VEGF) protein expression in cultures of primary human RPE (hRPE) cells and a human RPE cell line (ARPE-19). METHODS: Two in vitro methods, scratching and micropatterning, were used to control the physical dissociation of RPE cell-cell junctions. Scratching was performed by scoring monolayers of RPE cells with a cell scraper. Micropatterning was achieved by using a stencil patterning method. Extracellular VEGF expression was assessed by using an enzyme-linked immunosorbent assay (ELISA) kit. Immunocytochemistry (ICC) was performed to visualize the expression and localization of VEGF and intercellular proteins zonula occludens-1 (ZO-1), N-cadherin, ß-catenin, and claudin-1 in RPE cultures. RESULTS: Higher expression of VEGF protein by cells on the edges of the scratched RPE layers was confirmed with ICC in short-term (1 day after confluency) and long-term (4 weeks after confluency) cultures. According to the ICC results, ZO-1, N-cadherin, ß-catenin, and claudin-1 successfully localized to cell-cell junctions in long-term cultures of ARPE-19 and hRPE cells. However, unlike N-cadherin, ß-catenin, and claudin-1, only ZO-1 localized junctionally in short-term cultures of both cell types. Moreover, removing cell-cell junctions by scratching resulted in the delocalization of ZO-1 from tight junctions to the cytoplasm. The loss of tight junction formation and the accumulation of ZO-1 in the cytoplasm correlated with increased VEGF expression. Micropatterning RPE cells on different sized circular patterns produced varying concentrations of cells with lost cell-cell junctions. When fewer cells formed intercellular junctions, increased extracellular VEGF secretion was observed from the ARPE-19 and hRPE cells. CONCLUSIONS: VEGF expression increases after physical disruption of RPE cell-cell connections. This increase in VEGF expression correlates with the loss of intercellular junctions and the localization of ZO-1 in the cytoplasm of RPE cells.

Alternative cDEP Design to Facilitate Cell Isolation for Identification by Raman Spectroscopy.

Dielectrophoresis (DEP) uses non-uniform electric fields to cause motion in particles due to the particles' intrinsic properties. As such, DEP is a well-suited label-free means for cell sorting. Of the various methods of implementing DEP, contactless dielectrophoresis (cDEP) is advantageous as it avoids common problems associated with DEP, such as electrode fouling and electrolysis. Unfortunately, cDEP devices can be difficult to fabricate, replicate, and reuse. In addition, the operating parameters are limited by the dielectric breakdown of polydimethylsiloxane (PDMS). This study presents an alternative way to fabricate a cDEP device allowing for higher operating voltages, improved replication, and the opportunity for analysis using Raman spectroscopy. In this device, channels were formed in fused silica rather than PDMS. The device successfully trapped 3.3 µm polystyrene spheres for analysis by Raman spectroscopy. The successful implementation indicates the potential to use cDEP to isolate and identify biological samples on a single device.

Nanoparticle properties and synthesis effects on surface-enhanced Raman scattering enhancement factor: an introduction.

Raman spectroscopy has enabled researchers to map the specific chemical makeup of surfaces, solutions, and even cells. However, the inherent insensitivity of the technique makes it difficult to use and statistically complicated. When Raman active molecules are near gold or silver nanoparticles, the Raman intensity is significantly amplified. This phenomenon is referred to as surface-enhanced Raman spectroscopy (SERS). The extent of SERS enhancement is due to a variety of factors such as nanoparticle size, shape, material, and configuration. The choice of Raman reporters and protective coatings will also influence SERS enhancement. This review provides an introduction to how these factors influence signal enhancement and how to optimize them during synthesis of SERS nanoparticles.

Raman spectroscopy provides a noninvasive approach for determining biochemical composition of the pregnant cervix in vivo.

UNLABELLED: The molecular changes that occur with cervical remodelling during pregnancy are not completely understood. This study reviews Raman spectroscopy, an optical technique for detecting changes in the pregnant cervix, and reports preliminary studies on cervical remodelling in mice that suggest that the technique provides advantages over other methods. CONCLUSION: Raman spectroscopy is sensitive to biochemical changes in the pregnant cervix and has high potential as a tool for detecting premature cervical remodelling in pregnant women.

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Can Ganciclovir and Quercetin-P188 Ameliorate Cytomegalovirus Induced Hearing Loss?

OBJECTIVE: Determine whether combination therapy with ganciclovir (GCV) and a Quercetin-P188 solution improves hearing outcomes in a murine cytomegalovirus (CMV) model. METHODS: BALB/c mice were infected with murine CMV on postnatal day 3 (p3). Quercetin was solubilized in saline using P188 (QP188). Treatment groups received either GCV, QP188, GCV and QP188, or P188 delivery vehicle BID at 12-hour intervals via intraperitoneal injection. All treatment groups were treated for 14 days starting at p3. Uninfected controls were treated with the combined regimen, saline or P188 delivery vehicle. Auditory thresholds were assessed using distortion product otoacoustic emission (DPOAE) and auditory brainstem response (ABR) testing at 4, 6, and 8 weeks of age. Temporal bones from separate CMV-infected groups were harvested at p10, and viral load was determined by quantitative polymerase chain reaction. RESULTS: CMV-infected mice receiving combination therapy GCV+QP188 demonstrated statistically significant lower ABR (p < 0.001) and DPOAE thresholds (p < 0.001) compared with mice treated with GCV monotherapy, QP188 monotherapy, and P188 delivery vehicle at 4, 6, and 8 weeks of age. GCV+QP188 combination therapy, GCV monotherapy, and QP188 monotherapy resulted in a nonsignificant reduction in mean viral titers compared to P188 monotherapy (p = 0.08). CONCLUSION: Combining GCV with the excipients quercetin and P188 effectively ameliorated CMV-induced sensorineural hearing loss in a murine model. This result may be partially explained by a reduction in viral titers in mouse temporal bones that correlate with in vitro studies demonstrating additive antiviral effect in cell culture. LEVEL OF EVIDENCE: NA Laryngoscope, 134:1457-1463, 2024.

Engineering a Biomimetic In Vitro Model of Bruch's Membrane Using Hagfish Slime Intermediate Filament Proteins.

Bruch's membrane resides in the subretinal tissue and regulates the flow of nutrients and waste between the retinal pigment epithelial (RPE) and vascular layers of the eye. With age, Bruch's membrane becomes thicker, stiffer, and less permeable, which impedes its function as a boundary layer in the subretina. These changes contribute to pathologies such as age-related macular degeneration (AMD). To better understand how aging in Bruch's membrane affects surrounding tissues and to determine the relationship between aging and disease, an in vitro model of Bruch's membrane is needed. An accurate model of Bruch's membrane must be a proteinaceous, semipermeable, and nonporous biomaterial with similar mechanical properties to in vivo conditions. Additionally, this model must support RPE cell growth. While models of subretinal tissue exist, they typically differ from in vivo Bruch's membrane in one or more of these properties. This study evaluates the capability of membranes created from recombinant hagfish intermediate filament (rHIF) proteins to accurately replicate Bruch's membrane in an in vitro model of the subretinal tissue. The physical characteristics of these rHIF membranes were evaluated using mechanical testing, permeability assays, brightfield microscopy, and scanning electron microscopy. The capacity of the membranes to support RPE cell culture was determined using brightfield and fluorescent microscopy, as well as immunocytochemical staining. This study demonstrates that rHIF protein membranes are an appropriate biomaterial to accurately mimic both healthy and aged Bruch's membrane for in vitro modeling of the subretinal tissue.

Poloxamer 188 - quercetin formulations amplify in vitro ganciclovir antiviral activity against cytomegalovirus.

Treatment of human cytomegalovirus (CMV) infection requires long-term administration of nucleoside analog antivirals such as ganciclovir (GCV), a therapy frequently limited by GCV-induced toxicity. Here, combining GCV treatment with two bioactive excipients, poloxamer 188 and quercetin, was investigated in vitro to reduce GCV dosage. Quercetin is a natural flavonoid exhibiting antiviral activity against CMV by a mechanism distinct from GCV, but is poorly soluble, limiting its use as a therapeutic. To overcome this challenge, quercetin was co-formulated with poloxamer 188 (P188, Pluronic ® F68). Quercetin-P188 (QP188) formulations yielded only modest CMV viral inhibition, with a selectivity index of 11.4, contrasted with a GCV selectivity index of 95. More significantly, when coadministered with GCV, QP188 exhibited an additive or synergistic interaction in subtherapeutic ranges of GCV. Fluorescence microscopy revealed QP188 accumulation in fibroblast mitochondria, suggesting that the excipient may modulate mitochondrial processes relevant to CMV infection. GCV antiviral therapy augmented with poloxamer-solubilized quercetin may be a viable approach to maintain CMV inhibition while lowering GCV doses, translating to reduced associated toxicity.

Detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction.

In traditional immuno-polymerase chain reaction (immuno-PCR), a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. Here we describe a nanoparticle-amplified immuno-PCR (NPA-IPCR) assay that combines antibody recognition of enzyme-linked immunosorbent assay (ELISA) with a 50-fold nanoparticle valence amplification step prior to tag amplification by PCR. The assay detects a respiratory syncytial virus (RSV) surface protein using an antibody bound to a 15-nm gold nanoparticle cofunctionalized with thiolated DNA complementary to a hybridized 76-base tag DNA with a tag DNA/antibody ratio of 50:1. The presence of virus particles triggers the formation of a "sandwich" complex composed of the gold nanoparticle construct, virus, and an antibody-functionalized magnetic particle used for extraction. After extraction, DNA tags are released by heating to 95°C and detected via real-time PCR. The limit of detection of the assay was compared with ELISA and reversion transcription (RT) PCR using RSV-infected HEp-2 cell extracts. NPA-IPCR showed an approximately 4000-fold improvement in the limit of detection compared with ELISA and a 4-fold improvement compared with viral RNA extraction followed by traditional RT-PCR. NPA-IPCR offers a viable platform for the development of early-stage diagnostics requiring an exceptionally low limit of detection.

Effect of normal variations on disease classification of Raman spectra from cervical tissue.

In this paper, we examine how variations in normal tissue can influence disease classification of Raman spectra. Raman spectra from normal areas may be affected by previous disease or proximity to areas of dysplasia. Spectra were acquired in vivo from 172 patients and classified into five tissue categories: true normal (no history of disease), previous disease normal (history of disease, current normal diagnosis), adjacent normal (disease on cervix, spectra acquired from visually normal area), low grade, and high grade. Taking into account the various "normal" states of the tissue before statistical analysis led to a disease classification accuracy of 97%. These results indicate that abnormal changes significantly affect Raman spectra, even when areas are histopathologically normal. The sensitivity of Raman spectroscopy to subtle biochemical differences must be considered in order to successfully implement it in a clinical setting for diagnosing cervical dysplasia and cancer.

Acute mechanical stress in primary porcine RPE cells induces angiogenic factor expression and in vitro angiogenesis.

BACKGROUND: Choroidal neovascularization (CNV) is a major cause of blindness in patients with age-related macular degeneration. CNV is characterized by new blood vessel growth and subretinal fluid accumulation, which results in mechanical pressure on retinal pigment epithelial (RPE) cells. The overexpression of RPE-derived angiogenic factors plays an important role in inducing CNV. In this work, we investigated the effect of mechanical stress on the expression of angiogenic factors in porcine RPE cells and determined the impact of conditioned medium on in-vitro angiogenesis. RESULTS: The goal of this study was to determine whether low levels of acute mechanical stress during early CNV can induce the expression of angiogenic factors in RPE cells and accelerate angiogenesis. Using a novel device, acute mechanical stress was applied to primary porcine RPE cells and the resulting changes in the expression of major angiogenic factors, VEGF, ANG2, HIF-1α, IL6, IL8 and TNF-α, were examined using immunocytochemistry, qRT-PCR, and ELISA. An in vitro tube formation assay was used to determine the effect of secreted angiogenic proteins due to mechanical stress on endothelial tube formation by human umbilical vein endothelial cells (HUVECs). Our results showed an increase in the expression of VEGF, ANG2, IL-6 and IL-8 in response to mechanical stress, resulting in increased in vitro angiogenesis. Abnormal epithelial-mesenchymal transition (EMT) in RPE cells is also associated with CNV and further retinal degeneration. Our qRT-PCR results verified an increase in the expression of EMT genes, CDH2, VIM and FN1, in RPE cells. CONCLUSIONS: In conclusion, we showed that acute mechanical stress induces the expression of major angiogenic and EMT factors and promotes in vitro angiogenesis, suggesting that mechanical stress plays a role in promoting aberrant angiogenesis in AMD.

Abiotic stressors impact outer membrane vesicle composition in a beneficial rhizobacterium: Raman spectroscopy characterization.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have roles in cell-to-cell signaling, biofilm formation, and stress responses. Here, the effects of abiotic stressors on OMV contents and composition from biofilm cells of the plant health-promoting bacterium Pseudomonas chlororaphis O6 (PcO6) are examined. Two stressors relevant to this root-colonizing bacterium were examined: CuO nanoparticles (NPs)-a potential fertilizer and fungicide- and H2O2-released from roots during plant stress responses. Atomic force microscopy revealed 40-300 nm diameter OMVs from control and stressed biofilm cells. Raman spectroscopy with linear discriminant analysis (LDA) was used to identify changes in chemical profiles of PcO6 cells and resultant OMVs according to the cellular stressor with 84.7% and 83.3% accuracies, respectively. All OMVs had higher relative concentrations of proteins, lipids, and nucleic acids than PcO6 cells. The nucleic acid concentration in OMVs exhibited a cellular stressor-dependent increase: CuO NP-induced OMVs > H2O2-induced OMVs > control OMVs. Biochemical assays confirmed the presence of lipopolysaccharides, nucleic acids, and protein in OMVs; however, these assays did not discriminate OMV composition according to the cellular stressor. These results demonstrate the sensitivity of Raman spectroscopy using LDA to characterize and distinguish cellular stress effects on OMVs composition and contents.

Silkworm Silk Fiber Bundles as Improved In Vitro Scaffolds for Skeletal Muscle.

To mimic skeletal muscle tissues in vitro, native and transgenic spider silk/silkworm silks were seeded with C2C12 myoblasts to observe if these three-dimensional substrates are preferable to a traditional two-dimensional polystyrene cell culture surface. Silks were wound around an acrylic chassis to produce a novel, three-dimensional cell culture device with suspended muscle fibers that genetically and morphologically resemble native skeletal muscle tissue. The transgenic spider silk/silkworm silk has never before been studied for this application. Genetic expression verified skeletal muscle lineage and differentiation, while fluorescent imaging verified contractile protein synthesis. Genetic analysis also revealed an increase in expression of the Myh2 contractile protein gene on silkworm silks, particularly on the transgenic silk. Mechanical properties and protein secondary structure content of the silks indicated correlation between substrate properties and Myh2 gene expression. This increase in contractile protein gene expression suggests that biologically derived silk substrates that are suspended may be a preferable substrate for in vitro muscle modeling because of the proteinaceous character and mechanical flexibility of the silk.

Muscle Atrophy Marker Expression Differs between Rotary Cell Culture System and Animal Studies.

Muscular atrophy, defined as the loss of muscle tissue, is a serious issue for immobilized patients on Earth and for humans during spaceflight, where microgravity prevents normal muscle loading. In vitro modeling is an important step in understanding atrophy mechanisms and testing countermeasures before animal trials. The most ideal environment for modeling must be empirically determined to best mimic known responses in vivo. To simulate microgravity conditions, murine C2C12 myoblasts were cultured in a rotary cell culture system (RCCS). Alginate encapsulation was compared against polystyrene microcarrier beads as a substrate for culturing these adherent muscle cells. Changes after culture under simulated microgravity were characterized by assessing mRNA expression of MuRF1, MAFbx, Caspase 3, Akt2, mTOR, Ankrd1, and Foxo3. Protein concentration of myosin heavy chain 4 (Myh4) was used as a differentiation marker. Cell morphology and substrate structure were evaluated with brightfield and fluorescent imaging. Differentiated C2C12 cells encapsulated in alginate had a significant increase in MuRF1 only following simulated microgravity culture and were morphologically dissimilar to normal cultured muscle tissue. On the other hand, C2C12 cells cultured on polystyrene microcarriers had significantly increased expression of MuRF1, Caspase 3, and Foxo3 and easily identifiable multinucleated myotubes. The extent of differentiation was higher in simulated microgravity and protein synthesis more active with increased Myh4, Akt2, and mTOR. The in vitro microcarrier model described herein significantly increases expression of several of the same atrophy markers as in vivo models. However, unlike animal models, MAFbx and Ankrd1 were not significantly increased and the fold change in MuRF1 and Foxo3 was lower than expected. Using a standard commercially available RCCS, the substrates and culture methods described only partially model changes in mRNAs associated with atrophy in vivo.

Effect of growth media and phase on Raman spectra and discrimination of mycobacteria.

When developing a Raman spectral library to identify bacteria, differences between laboratory and real world conditions must be considered. For example, culturing bacteria in laboratory settings is performed under conditions for ideal bacteria growth. In contrast, culture conditions in the human body may differ and may not support optimized bacterial growth. To address these differences, researchers have studied the effect of conditions such as growth media and phase on Raman spectra. However, the majority of these studies focused on Gram-positive or Gram-negative bacteria. This article focuses on the influence of growth media and phase on Raman spectra and discrimination of mycobacteria, an acid-fast genus. Results showed that spectral differences from growth phase and media can be distinguished by spectral observation and multivariate analysis. Results were comparable to those found for other types of bacteria, such as Gram-positive and Gram-negative. In addition, the influence of growth phase and media had a significant impact on machine learning models and their resulting classification accuracy. This study highlights the need for machine learning models and their associated spectral libraries to account for various growth parameters and stages to further the transition of Raman spectral analysis of bacteria from laboratory to clinical settings.

Utilizing Recombinant Spider Silk Proteins To Develop a Synthetic Bruch's Membrane for Modeling the Retinal Pigment Epithelium.

Spider silks are intriguing biomaterials that have a high potential as innovative biomedical processes and devices. The intent of this study was to evaluate the capacity of recombinant spider silk proteins (rSSps) as a synthetic Bruch's membrane. Nonporous silk membranes were prepared with comparable thicknesses (<10 µm) to that of native Bruch's membrane. Biomechanical characterization was performed prior to seeding cells. The ability of RPE cells (ARPE-19) to attach and grow on the membranes was then evaluated with bright-field and electron microscopy, intracellular DNA quantification, and immunocytochemical staining (ZO-1 and F-actin). Controls were cultured on permeable Transwell support membranes and characterized with the same methods. A size-dependent permeability assay, using FITC-dextran, was used to determine cell-membrane barrier function. Compared to Transwell controls, RPE cells cultured on rSSps membranes developed more native-like "cobblestone" morphologies, exhibited higher intracellular DNA content, and expressed key organizational proteins more consistently. Comparisons of the membranes to native structures revealed that the silk membranes exhibited equivalent thicknesses, biomechanical properties, and barrier functions. These findings support the use of recombinant spider silk proteins to model Bruch's membrane and develop more biomimetic retinal models.

Effect of c-neu/ ErbB2 expression levels on estrogen receptor alpha-dependent proliferation in mammary epithelial cells: implications for breast cancer biology.

Mammary development and tumorigenesis are profoundly influenced by signaling pathways under the control of c-erbB2/c-neu and estrogen receptor alpha (ERalpha). Signaling through ERalpha is essential for ductal growth during puberty. In mice overexpressing wild-type c-neu in mammary epithelial cells, Tg (c-neu), ductal growth is impaired. An impeded signaling through ERalpha is also observed in a subset of human mammary tumors that overexpress erbB2. However, ductal growth is also impaired in the absence of c-neu in mouse mammary epithelial cells. To resolve this apparent paradox, we examined the relationship between c-neu expression and estrogen/ERalpha-dependent cell proliferation in pubertal Tg (c-neu). We report that proliferation in both terminal end buds and ducts is associated with ERalpha-positive cells, including those that coexpress c-neu, and is abolished in the absence of circulating estradiol. Tg (c-neu) contains hyperplastic mammary ducts with high proliferative index and coexpression of both ERalpha and c-neu in the dividing cells. These findings suggest that c-neu promotes ERalpha-dependent proliferation, and that this is responsible for the presence of hyperplastic ducts. Some of the hyperplastic ducts have acinar structures, indicative of morphologic differentiation. These ducts have low proliferative index and accompanied by a vast decrease in proliferation of ERalpha-positive cells, including those that express c-neu. As such, c-neu has dual but opposing effects on ERalpha-dependent proliferation in mammary epithelial cells. Therefore, depending on the physiologic setting, ductal morphogenesis will be compromised both in the absence and overexpression of c-neu, thus explaining the paradox.