Cell Phase Identification in a Three-Dimensional Engineered Tumor Model by Infrared Spectroscopic Imaging.

Cell cycle progression plays a vital role in regulating proliferation, metabolism, and apoptosis. Three-dimensional (3D) cell cultures have emerged as an important class of in vitro disease models, and incorporating the variation occurring from cell cycle progression in these systems is critical. Here, we report the use of Fourier transform infrared (FT-IR) spectroscopic imaging to identify subtle biochemical changes within cells, indicative of the G1/S and G2/M phases of the cell cycle. Following previous studies, we first synchronized samples from two-dimensional (2D) cell cultures, confirmed their states by flow cytometry and DNA quantification, and recorded spectra. We determined two critical wavenumbers (1059 and 1219 cm-1) as spectral indicators of the cell cycle for a set of isogenic breast cancer cell lines (MCF10AT series). These two simple spectral markers were then applied to distinguish cell cycle stages in a 3D cell culture model using four cell lines that represent the main stages of cancer progression from normal cells to metastatic disease. Temporal dependence of spectral biomarkers during acini maturation validated the hypothesis that the cells are more proliferative in the early stages of acini development; later stages of the culture showed stability in the overall composition but unique spatial differences in cells in the two phases. Altogether, this study presents a computational and quantitative approach for cell phase analysis in tissue-like 3D structures without any biomarker staining and provides a means to characterize the impact of the cell cycle on 3D biological systems and disease diagnostic studies using IR imaging.

Psychological impact, coping behaviors, and traumatic stress among healthcare workers during COVID-19 in Taiwan: An early stage experience.

OBJECTIVE: This study investigated the psychological impact on, coping behaviors of, and traumatic stress experienced by healthcare workers during the early stage of the COVID-19 pandemic and formulated effective support strategies that can be implemented by hospitals and government policymakers to help healthcare staff overcome the pandemic. METHODS: This cross-sectional study recruited clinical healthcare workers at a regional hospital in Nantou County, Taiwan. The questionnaire collected personal characteristics, data on the impact and coping behaviors of the pandemic, and Impact of Event Scale-Revised (IES-R). A total of 354 valid questionnaires were collected. The statistical methods employed were univariate and multivariate stepwise regression, and logistic regression. RESULTS: Perceived impact and coping behaviors were found to be moderate in degree, and traumatic stress was lower than that in other countries. However, our data identified the following subgroups that require special attention: those with young age, those living with minor children, nurses, those with self-rated poor mental health, and those with insufficient COVID-19-related training. CONCLUSION: Managers should pay particular attention to helping healthcare workers in high-risk groups, strengthen COVID-19 training, provide adequate protective equipment and shelter, and offer psychological counseling.

On the Limit of Detection in Infrared Spectroscopic Imaging.

Infrared (IR) spectroscopic imaging instruments' performance can be characterized and optimized by an analysis of their limit of detection (LOD). Here we report a systematic analysis of the LOD for Fourier transform IR (FT-IR) and discrete frequency IR (DFIR) imaging spectrometers. In addition to traditional measurements of sample and blank data, we propose a decision theory perspective to pose the determination of LOD as a binary classification problem under different assumptions of noise uniformity and correlation. We also examine three spectral analysis approaches, namely, absorbance at a single frequency, average of absorbance over selected frequencies and total spectral distance - to suit instruments that acquire discrete or contiguous spectral bandwidths. The analysis is validated by refining the fabrication of a bovine serum albumin protein microarray to provide eight uniform spots from ∼2.8 nL of solution for each concentration over a wide range (0.05-10 mg/mL). Using scanning parameters that are typical for each instrument, we estimate a LOD of 0.16 mg/mL and 0.12 mg/mL for widefield and line scanning FT-IR imaging systems, respectively, using the spectral distance approach, and 0.22 mg/mL and 0.15 mg/mL using an optimal set of discrete frequencies. As expected, averaging and the use of post-processing techniques such as minimum noise fraction transformation results in LODs as low as ∼0.075 mg/mL that correspond to a spotted protein mass of ∼112 fg/pixel. We emphasize that these measurements were conducted at typical imaging parameters for each instrument and can be improved using the usual trading rules of IR spectroscopy. This systematic analysis and methodology for determining the LOD can allow for quantitative measures of confidence in imaging an analyte's concentration and a basis for further improving IR imaging technology.

Freeform Three-Dimensionally Printed Microchannels via Surface-Initiated Photopolymerization Combined with Sacrificial Molding.

Precise freeform microchannels within an aqueous environment have several biomedical applications but remain a challenge to fabricate. Carbohydrate glass materials have shown potential for three-dimensionally (3D) printing precise, microscale structures and are suitable as a sacrificial material to reconstruct complex channel architectures, but due to the rapid dissolution kinetics in hydrogels and the aqueous environment, protective coatings are required. Here, conformal coatings were applied to carbohydrate structures via surface-initiated photopolymerization (SIP) by incorporating a photoinitiator (PI) into freeform 3D printed isomalt structures using a custom 3D printer. Structures were then immersed into a photocurable prepolymer bath and exposed to light for reaction initiation. To achieve uniform distribution of photoinitiator molecules in 3D printed constructs, miscibility between commercial photoinitiators and isomalt was modeled using the group contribution method. A dye-based, type-two photoinitiator, Eosin Y disodium salt (EY), was selected for its miscibility with isomalt and stability under high temperature. A previously described Eosin Y (EY)/triethanolamine (TEA) radical polymerization system was used to polymerize poly(ethylene glycol) diacrylate (PEGDA). Attenuated total reflectance-Fourier transform infrared (ATR-FTIR), surface morphology, and swelling ratio characterizations via SIP were performed. Coatings around freeform structures and solid surfaces were presented to demonstrate the capability of coating complex architectures. This coating method should facilitate the application of 3D sacrificial molding in a variety of hydrogels toward building biomimetic vascular constructs.

Bioinspired Self-assembling Peptide Hydrogel with Proteoglycan-assisted Growth Factor Delivery for Therapeutic Angiogenesis.

Critical challenges still exist in surgical theaters and emergency rooms to stop bleeding effectively and facilitate wound healing efficiently. In circumstances of tissue ischemia, it is essential to induce proper angiogenesis to provide adequate vascular supply to the injury site. Methods: In view of these clinical unmet needs, we propose an applicable approach by designing functionalized self-assembling peptide (SAP) hydrogel with two sequences of RADA16-GGQQLK (QLK) and RADA16-GGLRKKLGKA (LRK) in this study. The SAP hydrogel conjugated with QLK functional motif could be crosslinked by endogenous transglutaminase, one of the intrinsic factors secreted during the coagulation process, the mechanical property of the hydrogel can then be enhanced without the need of external support. On the other hand, the LRK sequence exhibited a good binding affinity with the proteoglycan heparan sulfate and could act as a cofactor by sustaining the release of embedded growth factors. Results: The results showed that this SAP solution underwent self-assembling process in a physiological environment, formed hydrogel in situ, and possessed good shear thinning property with injectability. After pH adjustment, the SAP developed densely-compacted fiber entanglement that closely mimicked the three-dimensional fibrous framework of natural extracellular matrix. Such scaffold could not only support the survival of encapsulating cells but also promote the capillary-like tubular structure formation by dual angiogenic growth factors. The ex ovo chicken chorioallantoic membrane assay demonstrated that the growth factor-loaded hydrogel promoted the sprout of surrounding vessels in a spoke-wheel pattern compared to growth factor-free counterparts. Conclusion: The designer bioinspired SAP hydrogel may be an attractive and promising therapeutic modality for minimally-invasive surgery, ischemic tissue disorders and chronic wound healing.

Cellular models for the evaluation of the antiobesity effect of selected phytochemicals from food and herbs.

Dietary phytochemicals from food and herbs have been studied for their health benefits for a long time. The incidence of obesity has seen an incredible increase worldwide. Although dieting, along with increased physical activity, seems an easy method in theory to manage obesity, it is hard to apply in real life. Obesity treatment drugs and surgery are not successful or targeted for everyone and can have significant side effects. This low rate of success is the major reason that the overweight as well as the pharmaceutical industry seek alternative methods, including phytochemicals. Therefore, more and more research has focused on the role of phytochemicals to alleviate lipid accumulation or enhance energy expenditure in adipocytes. This review discusses selected phytochemicals from food and herbs and their effects on adipogenesis, lipogenesis, lipolysis, oxidation of fatty acids, and browning in 3T3-L1 preadipocytes.

Streamlined Membrane Proteome Preparation for Shotgun Proteomics Analysis with Triton X-100 Cloud Point Extraction and Nanodiamond Solid Phase Extraction.

While mass spectrometry (MS) plays a key role in proteomics research, characterization of membrane proteins (MP) by MS has been a challenging task because of the presence of a host of interfering chemicals in the hydrophobic protein extraction process, and the low protease digestion efficiency. We report a sample preparation protocol, two-phase separation with Triton X-100, induced by NaCl, with coomassie blue added for visualizing the detergent-rich phase, which streamlines MP preparation for SDS-PAGE analysis of intact MP and shot-gun proteomic analyses. MP solubilized in the detergent-rich milieu were then sequentially extracted and fractionated by surface-oxidized nanodiamond (ND) at three pHs. The high MP affinity of ND enabled extensive washes for removal of salts, detergents, lipids, and other impurities to ensure uncompromised ensuing purposes, notably enhanced proteolytic digestion and down-stream mass spectrometric (MS) analyses. Starting with a typical membranous cellular lysate fraction harvested with centrifugation/ultracentrifugation, MP purities of 70%, based on number (not weight) of proteins identified by MS, was achieved; the weight-based purity can be expected to be much higher.

Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice.

Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA) is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD), a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR) injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877), 1.71% (15/877), and 2.62% (23/877) of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood-brain barrier (BBB) (Alb, Fga, and Trf), suppressed excitotoxicity (Grm5, Gnai, and Gdi), and enhanced energy metabolism (Bdh), thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3) and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke.

Outcomes of a rotational dissection system in gross anatomy.

At the University of Texas Houston Medical School, a rotational dissection system was introduced to improve coordination between the Gross Anatomy and the Introduction to Clinical Medicine (ICM) courses. Six students were assigned to each cadaver and divided into two teams. For each laboratory, one team was assigned to dissect and the other to attend ICM or study independently. For the next laboratory, the assignments were reversed. At the start of the session, the team that had dissected previously spent 30 minutes teaching the other team. In 2012, the students were given three traditional practical examinations with 50 questions drawn equally from each laboratory. Students also completed three mid-course evaluations. There were no significant differences in overall performance between the two teams. Nevertheless, we wanted to determine how well individual students identified structures they had dissected compared with those they had not. For dissected structures, the mean percent correct was 80.0 ± 13.0 (mean ± standard deviation), and for undissected structures, it was 78.3 ± 14.1. The difference was small, but statistically significant (P = 0.0007). Although this result validated the concerns expressed by some students, it did not appear that a change in the system was justified. Students were generally enthusiastic about the opportunity to learn clinical skills in the first semester of medical school, and 91-96% of the students agreed that learning anatomy at the same time helped them understand the physical examination exercises in ICM.

Junk food seen at pediatric clinic visits: is it a problem?

OBJECTIVE: To document the prevalence of junk foods seen at clinic visits. DESIGN: A cross-sectional 23-item survey of observed food items were completed by medical staff using a convenience sample of families from June 2, 2011 to March 2, 2012. SETTING: The study was conducted in pediatric clinics affiliated with the University of Texas Medical School at Houston. PARTICIPANTS: A convenience sample consisting of 738 families with children from 4 months to 16 years old presenting for visits were included in the study. Children exclusively breast and formula fed was excluded. RESULTS: Junk food was observed 20.9% at the clinic visits. CONCLUSIONS AND RELEVANCE: Junk food was often seen at clinic visits. There was a trend toward higher body mass index in patients whose families had junk food at the visit.

A maternal high n-6 fat diet with fish oil supplementation during pregnancy and lactation in rats decreases breast cancer risk in the female offspring.

The timing of dietary fat intake may modify breast cancer risk. In addition, n-3 fatty acids reduce, and n-6 fatty acids increase, the risk of breast cancer and a maternal high n-6 fat diet results in a greater risk of breast cancer in the female offspring. We hypothesized that the timing of n-3 fatty acid-enriched fish oil supplementation would be important for reducing the risk of breast cancer. Female rats were fed to a high n-6 fat diet containing 20% of the sunflower oil by weight during pregnancy and lactation, and the female offspring were exposed to fish oil by oral gavage either during the perinatal period via maternal intake or during puberty or adulthood. Exposure during the perinatal period to a maternal high n-6 fat diet with fish oil supplementation significantly reduced the incidence of carcinogen-induced mammary tumors in the female offspring compared to a maternal high n-6 fat diet with no fish oil supplementation or fish oil supplementation later in life (P=.0228 by Cox proportional hazards model). We found that a maternal high n-6 fat diet during pregnancy is more important in increasing the risk of mammary tumors in the female offspring than a maternal high n-6 fat diet during lactation. This study suggests that fish oil supplementation during the perinatal period decreases the effect of a maternal high n-6 fat diet on subsequent carcinogen-induced mammary tumor risk, whereas fish oil supplementation during puberty or adulthood does not.

A maternal high-fat diet during pregnancy in rats results in a greater risk of carcinogen-induced mammary tumors in the female offspring than exposure to a high-fat diet in postnatal life.

The association between a high-fat diet and breast cancer risk is controversial. We hypothesized that the exposure of rats to a high-fat diet in utero via the maternal diet would result in a greater risk of carcinogen-induced mammary tumors than high-fat diet exposure in postnatal life. Rats were exposed to a high-fat diet with 40% of the energy source as safflower oil in utero (In utero group), at postnatal days 30-50 (Puberty group), postnatal days 150-170 (Adult group), postnatal days 1-230 (Postnatal group) or for their whole life from in utero (Whole group). Chow diet-fed rats were used as the Nonexposure group. Mammary tumor incidence was significantly higher in the In utero (60%), Postnatal (61%) and Whole (91%) groups than in the Nonexposure group (32%), but there was no significant difference between the Puberty (44%), Adult (44%) and Nonexposure groups. Arachidonic acid levels were 10 times higher in mammary tumor tissue than in the normal mammary gland across all groups and were positively correlated with tumor weight. We conclude that the timing, but not the duration, of high-fat diet exposure makes rats more susceptible to carcinogen-induced mammary tumors and that exposure in utero to a maternal high-fat diet during pregnancy is more important in increasing the risk of mammary tumors in the female offspring than exposure of the offspring to the same high-fat diet later in life.