Chemical Milieu Research Articles

Chronic Exposure to Petroleum-Derived Hydrocarbons Alters Human Skin Microbiome and Metabolome Profiles: A Pilot Study.

Petroleum-derived substances, like industrial oils and grease, are ubiquitous in our daily lives. Comprised of petroleum hydrocarbons (PH), these substances can come into contact with our skin, potentially causing molecular disruptions and contributing to the development of chronic disease. In this pilot study, we employed mass spectrometry-based untargeted metabolomics and 16S rRNA gene sequencing analyses to explore these effects. Superficial skin samples were collected from subjects with and without chronic dermal exposure to PH at two anatomical sites: the fingers (referred to as the hand) and arms (serving as an intersubject variability control). Exposed hands exhibited higher bacterial diversity (Shannon and Simpson indices) and an enrichment of oil-degrading bacteria (ODB), including Dietzia, Paracoccus, and Kocuria. Functional prediction suggested enriched pathways associated with PH degradation in exposed hands vs non-exposed hands, while no differences were observed when comparing the arms. Furthermore, carboxylic acids, glycerophospholipids, organooxygen compounds, phenol ethers, among others, were found to be more abundant in exposed hands. We observed positive correlations among multiple ODB and xenobiotics, suggesting a chemical remodeling of the skin favorable for ODB thriving. Overall, our study offers insights into the complex dysregulation of bacterial communities and the chemical milieu induced by chronic dermal exposure to PH.

Alternative Pathways in Astrobiology: Reviewing and Synthesizing Contingency and Non-Biomolecular Origins of Terrestrial and Extraterrestrial Life.

The pursuit of understanding the origins of life (OoL) on and off Earth and the search for extraterrestrial life (ET) are central aspects of astrobiology. Despite the considerable efforts in both areas, more novel and multifaceted approaches are needed to address these profound questions with greater detail and with certainty. The complexity of the chemical milieu within ancient geological environments presents a diverse landscape where biomolecules and non-biomolecules interact. This interaction could lead to life as we know it, dominated by biomolecules, or to alternative forms of life where non-biomolecules could play a pivotal role. Such alternative forms of life could be found beyond Earth, i.e., on exoplanets and the moons of Jupiter and Saturn. Challenging the notion that all life, including ET life, must use the same building blocks as life on Earth, the concept of contingency-when expanded beyond its macroevolution interpretation-suggests that non-biomolecules may have played essential roles at the OoL. Here, we review the possible role of contingency and non-biomolecules at the OoL and synthesize a conceptual model formally linking contingency with non-biomolecular OoL theories. This model emphasizes the significance of considering the role of non-biomolecules both at the OoL on Earth or beyond, as well as their potential as agnostic biosignatures indicative of ET Life.

Rebuilding the Habitable Zone from the Bottom up with Computational Zones.

Computation, if treated as a set of physical processes that act on information represented by states of matter, encompasses biological systems, digital systems, and other constructs and may be a fundamental measure of living systems. The opportunity for biological computation, represented in the propagation and selection-driven evolution of information-carrying organic molecular structures, has been partially characterized in terms of planetary habitable zones (HZs) based on primary conditions such as temperature and the presence of liquid water. A generalization of this concept to computational zones (CZs) is proposed, with constraints set by three principal characteristics: capacity (including computation rates), energy, and instantiation (or substrate, including spatial extent). CZs naturally combine traditional habitability factors, including those associated with biological function that incorporate the chemical milieu, constraints on nutrients and free energy, as well as element availability. Two example applications are presented by examining the fundamental thermodynamic work efficiency and Landauer limit of photon-driven biological computation on planetary surfaces and of generalized computation in stellar energy capture structures (a.k.a. Dyson structures). It is suggested that CZs that involve nested structures or substellar objects could manifest unique observational signatures as cool far-infrared emitters. While these latter scenarios are entirely hypothetical, they offer a useful, complementary introduction to the potential universality of CZs.

An analytical evaluation of tools for lipid isomer differentiation in imaging mass spectrometry

Imaging mass spectrometry has emerged as a powerful tool to map the spatial distributions of lipids and metabolites in biological tissues. However, these analyses are challenged by the multitude of isobaric (i.e., same nominal mass) and isomeric compounds present in most samples. Failure to adequately separate these compounds results in inaccurate or incomplete chemical identifications and produces composite images of spatial distribution arising from multiple compounds. A number of techniques have been developed to more completely resolve and identify this complex chemical milieu. These include methods that rely on condensed-phase chemical derivatization and gas-phase ion chemistry, or some combination thereof. This Young Scientist Feature focuses on summarizing the analytical figures of merit of these tools, highlighting their relative speeds, limits of detection, molecular specificities, and eases-of-use. It will also include current challenges and future perspectives for resolving structural isomers in imaging mass spectrometry experiments.

VCP/p97 mediates nuclear targeting of non-ER-imported prion protein to maintain proteostasis.

Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding.

Review of Gold Nanoparticles in Surface Plasmon-Coupled Emission Technology: Effect of Shape, Hollow Nanostructures, Nano-Assembly, Metal-Dielectric and Heterometallic Nanohybrids.

Point-of-care (POC) diagnostic platforms are globally employed in modern smart technologies to detect events or changes in the analyte concentration and provide qualitative and quantitative information in biosensing. Surface plasmon-coupled emission (SPCE) technology has emerged as an effective POC diagnostic tool for developing robust biosensing frameworks. The simplicity, robustness and relevance of the technology has attracted researchers in physical, chemical and biological milieu on account of its unique attributes such as high specificity, sensitivity, low background noise, highly polarized, sharply directional, excellent spectral resolution capabilities. In the past decade, numerous nano-fabrication methods have been developed for augmenting the performance of the conventional SPCE technology. Among them the utility of plasmonic gold nanoparticles (AuNPs) has enabled the demonstration of plethora of reliable biosensing platforms. Here, we review the nano-engineering and biosensing applications of AuNPs based on the shape, hollow morphology, metal-dielectric, nano-assembly and heterometallic nanohybrids under optical as well as biosensing competencies. The current review emphasizes the recent past and evaluates the latest advancements in the field to comprehend the futuristic scope and perspectives of exploiting Au nano-antennas for plasmonic hotspot generation in SPCE technology.

CNSC-29. INVESTIGATING THE INFLUENCE OF DOPAMINERGIC ACTIVITY ON THE GLIOBLASTOMA NICHE

Abstract Glioblastoma (GBM) is an incurable disease of adults and children and the deadliest form of central nervous system (CNS) malignancy. Despite major advances in our understanding of GBM biology in recent years, the prognosis for patients who develop this disease has not improved. If we hope to find new treatments for GBM that are safe and effective, we desperately need to reform our thinking. The brain’s chemical milieu is rich with neurotransmitters, and our lab’s screen of 680 neuroactive compounds on patient-derived glioblastoma stem cells (GSCs) in vitro strongly implicates neurotransmitter pathways as critical regulators of the GSC niche. More specifically, disrupting dopamine signaling by inhibiting its receptor D4 on GSCs causes substantial GSC apoptosis in vitro and attenuates GBM growth in vivo (Dolma et al., Cancer Cell, 2016). Dopamine (DA) is a catecholamine neurotransmitter that is essential for reward learning and movement and has numerous roles in cognition. Consequentially, dysregulation of DA signaling is associated with a diversity of brain diseases ranging from drug addiction to schizophrenia to Parkinson’s. Our research aims to determine how DA signaling affects normal neural stem cell (NSC) and tumorigenic GSC populations, as we hypothesize that GSCs arising/residing in DA projection zones exploit dopaminergic (DAergic) activity for GBM growth. Toward these aims, we have developed in vivo model systems and harnessed them to study NSC and GSC niches in the context of either controlled activation or depletion of DAergic neurons. These manipulations of the brain's DAergic neurons are achieved using optogenetic stimulation, genetic depletion, and neurotoxin-mediated ablation in mice. Ultimately, unraveling the dopaminergic influence on GBM may contribute to a redeployment of existing treatments—that modulate DA signaling and are already approved to treat CNS disorders—to patients with this deadly brain cancer.

Effects of physico-chemical treatments on PLGA 50:50 electrospun nanofibers

Biocompatible polymers are used as scaffolds to mimic the native extracellular matrix (ECM) in tissue engineering applications. However, when integrated into microengineered cell and tissue cultures, such scaffolds are often subjected to several alterations in the chemical milieu for drug testing purposes, modelling a disease, etc. Changes in the chemical environment may affect the architecture and function of the engineered scaffolds; therefore, it is important to study the material and structural changes that can occur in the scaffolds and the stimuli that bring about those changes. Nanofiber membranes of poly(D, L-lactic-co-glycolic acid) (PLGA) (Lactic acid Glycolic acid 50/50) were subjected to thermal treatments in air and thermo-chemical treatments in deionized water, ethanol and 2-Propanol at various temperatures to study structural changes due to swelling. The effects of the physico-chemical treatments on the nanofiber films were quantified by studying the average fiber diameter and porosity. Physico-chemical treatments led to the creation of interfiber bonds in the nanofibers. Interfiber bonds are believed to be formed by the fusion of mobile macromolecular polymer chains in the fibers due to increased Brownian motion. Rates of increase of average fiber diameter were 6.6 ± 1.2 to 26 ± 10 times faster in thermochemical treatments than in air at 50 °C. This is believed to be caused by a coupled thermo-chemical effect due to physico-chemical interactions between the reagent molecules and the nanofibers. Glass-transition temperatures of the nanofiber films showed a 23.5 ± 1.8% decrease due to solvation in water and a 12.6 ± 3.7% increase due to solvation in ethanol at room temperature, respectively. Simple physico-chemical treatments like heat and solvation have been shown to change the structural (fiber diameter, porosity) and material (thermal) properties of PLGA nanofiber scaffolds. Such changes are important to study and characterize, especially for biomedical applications of nanofiber scaffolds.

Elevated 18:0 lysophosphatidylcholine contributes to the development of pain in tissue injury.

Tissue injuries, including burns, are major causes of death and morbidity worldwide. These injuries result in the release of intracellular molecules and subsequent inflammatory reactions, changing the tissues' chemical milieu and leading to the development of persistent pain through activating pain-sensing primary sensory neurons. However, the majority of pain-inducing agents in injured tissues are unknown. Here, we report that, amongst other important metabolite changes, lysophosphatidylcholines (LPCs) including 18:0 LPC exhibit significant and consistent local burn injury-induced changes in concentration. 18:0 LPC induces immediate pain and the development of hypersensitivities to mechanical and heat stimuli through molecules including the transient receptor potential ion channel, vanilloid subfamily, member 1, and member 2 at least partly via increasing lateral pressure in the membrane. As levels of LPCs including 18:0 LPC increase in other tissue injuries, our data reveal a novel role for these lipids in injury-associated pain. These findings have high potential to improve patient care.

Nitinol Release of Nickel under Physiological Conditions: Effects of Surface Oxide, pH, Hydrogen Peroxide, and Sodium Hypochlorite.

Nitinol is a nickel-titanium alloy widely used in medical devices for its unique pseudoelastic and shape-memory properties. However, nitinol can release potentially hazardous amounts of nickel, depending on surface manufacturing yielding different oxide thicknesses and compositions. Furthermore, nitinol medical devices can be implanted throughout the body and exposed to extremes in pH and reactive oxygen species (ROS), but few tools exist for evaluating nickel release under such physiological conditions. Even in cardiovascular applications, where nitinol medical devices are relatively common and the blood environment is well understood, there is a lack of information on how local inflammatory conditions after implantation might affect nickel ion release. For this study, nickel release from nitinol wires of different finishes was measured in pH conditions and at ROS concentrations selected to encompass and exceed literature reports of extracellular pH and ROS. Results showed increased nickel release at levels of pH and ROS reported to be physiological, with decreasing pH and increasing concentrations of hydrogen peroxide and NaOCl/HOCl having the greatest effects. The results support the importance of considering the implantation site when designing studies to predict nickel release from nitinol and underscore the value of understanding the chemical milieu at the device-tissue interface.

Abstract P1-08-31: Simbiosys tumorscope: Biophysical modeling of patient-specific response to chemotherapy

Abstract Background: Breast Cancer (BC) patients exhibit a wide variety of responses to neoadjuvant chemotherapy (NACT). This is driven by factors both intrinsic (e.g., mutations, dysregulation, metabolic reprogramming) and extrinsic (e.g., nutrient/drug perfusion, interactions with surrounding healthy tissues and the tumor microenvironment (TME)) to the cells that make up each tumor. The SimBioSys TumorScope is a platform for making individualized predictions of the response of each patient's tumor to NACT. It employs 3D biophysical simulations that explicitly model the dynamics of cellular response to the ever-changing chemical milieu of drugs and nutrients that perfuse the TME during treatment, in order to predict when and where different regions of the tumor are growing, dying, and ultimately how a given patient will respond to treatment. Methods: The SimBioSys TumorScope constructs 3D in silico models of each patient's tumor directly from pretreatment DCE-MRIs. It combines this spatial model with personalized genome-scale models of tumor and tissue metabolism, pharmacokinetics, and pharmacodynamics, and vascular perfusion (based on DCE-MRI timeseries). The combined model is then simulated using a custom high-performance reaction-diffusion-material mechanics simulation engine which produces a spatio-temporal trajectory of tumor size, morphology, intra- and extracellular biochemistry. We evaluated the ability of the TumorScope software to predict volumetric response to NACT. A validation set comprising the pretreatment records (including MRIs) of 780 BC patients that underwent NACT was used. These patients spanned a wide range of tumor sizes, molecular subtypes, and NCCN-recommended treatment regimens. Simulations were initialized using each patient’s pretreatment MRI and pathology data and run from the start of therapy to the specified surgical date. Simulated tumor volumetric percent response (calculated as the ratio of change in tumor volume to initial volume) at the time of surgery was then compared with actual tumor volumetric percent response extracted from presurgical MRIs. Among patients for which event free survival data was available (n = 480), we performed a Cox proportional hazard analysis. Results: The SimBioSys TumorScope predicted pre-surgical tumor volumetric response with a median error of 0.03% and median absolute deviation of 8.2%. Among the patients for which EFS data was available, we found a hazard ratio of 1.8 associated with having a final simulated volume greater than 0.01 cc (p = 0.00048). Conclusions: The SimBioSys TumorScope produces accurate patient specific predictions of response to NACT using only standard-of-care pre-treatment data. Such predictions can aid in decision making, enabling physicians to select less-toxic regimens for patients in which a robust response is predicted, and more aggressive treatments and/or clinical trial enrollment when response is likely to be poor. Citation Format: John A Cole, Jr., Joseph R Peterson, Tyler M Earnest, Michael J Hallock, Daniel J Cook, Eduardo Braun, Anu Antony, John R Pfeiffer, Tushar Pandey. Simbiosys tumorscope: Biophysical modeling of patient-specific response to chemotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-31.

116 Weak Links in the Permeability Barrier of the Skin

The microbiota composition and chemical milieu have been shown to prominently differ in topographically distinct healthy skin areas, and recently, our workgroup identified differences in the immune activity of distinct regions as well. As a continuation of our work, we aimed to study the barrier function and compare the expression levels of major permeability barrier components (stratum corneum and tight junction [TJ]) at the molecular level in healthy sebaceous gland-rich (SGR), apocrine gland-rich (AGR), and gland-poor (GP) skin regions. Molecules involved in cornified envelope (CE) formation, desquamation and desmosome formation were investigated in parallel with TJ molecules at the mRNA and protein levels by qRT-PCR and immunohistochemistry. Desmosome and TJ structures were also visualized and quantified by confocal microscopy. While CE structure components were similarly expressed, kallikrein proteases showed significantly higher mRNA levels in SGR and AGR regions compared to GP, and immunohistochemistry indicated their prominent presence in sebaceous and apocrine glands. Desmosome and TJ components’ mRNA levels were similar in the three regions, in contrast, significantly lower protein levels of DSG1, CDSN (desmosome), and OCLN (TJ) molecule were detected in the SGR region, while in AGR skin, the presence of DSG1 (desmosome), OCLN and CLDN1 (TJ) was significantly decreased compared to GP areas. We propose that the increased levels of KLK enzymes and their degradation processes are responsible for the weakened cell junctions in SGR and AGR skin which may explain why certain acantholytic skin disorders (e.g. Darier’s disease, Hailey-Hailey disease) are localized to these areas.

193 Different Skin Regions are Characterized by Distinct Epimmunome Patterns

In recent years, we provided the first evidence that in parallel with their distinct microbial and chemical milieu, topographically distinct skin areas show different immune tuning. In the skin, keratinocytes (KCs) produce epimmunomes which molecules can instruct the innate and adaptive immune cells under both homeostatic and inflammatory conditions. In our current study, we aimed to investigate at the molecular level whether epimmunome molecules (IL-25, IL-33, IL-23, IL-8, IL-18, IL-6, IL-24, IL-17C, IL-1α, IL-1β, IL-36, IL-37, IL-38) show different expression levels in distinct healthy skin regions, namely in gland poor (GP), sebaceous gland rich (SGR), and apocrine gland rich (AGR) areas. The expression of epimmunomes were analyzed both at the mRNA and protein levels by RT-qPCR and IHC. According to our results, GP skin is characterized by a pro-Th2 epimmunome milieu with a significantly elevated expression of IL-25, IL-33 at the protein and gene level, while the IL-18, IL-36RA, IL-37 and IL-38 molecules were upregulated at the gene level compared to SGR and AGR regions. On the other hand, both SGR and AGR areas were characterized by a pro-Th17 epimmunome milieu with a significantly higher expression level of IL-17C protein, whereas, in SGR region, IL-1α was upregulated at the protein level while IL-1β and IL-8 at the mRNA level. Our results indicate that distinct skin regions have a unique epimmunome composition even under steady-state. Our findings may further explain why some outside-in skin diseases prefer to be localized to certain skin regions. We propose that the non-inflammatory pro-Th2 immune milieu of GP area predisposes it for the development of Th2-driven atopic dermatitis. On the contrary, the homeostatic pro-Th17 immune milieu makes AGR and SGR areas susceptible for the development of their characteristic Th1/Th17-driven region-specific diseases, hidradenitis suppurativa and rosacea, respectively.

Regional Differences in the Permeability Barrier of the Skin-Implications in Acantholytic Skin Diseases.

The chemical milieu, microbiota composition, and immune activity show prominent differences in distinct healthy skin areas. The objective of the current study was to compare the major permeability barrier components (stratum corneum and tight junction (TJ)), investigate the distribution of (corneo)desmosomes and TJs, and measure barrier function in healthy sebaceous gland-rich (SGR), apocrine gland-rich (AGR), and gland-poor (GP) skin regions. Molecules involved in cornified envelope (CE) formation, desquamation, and (corneo)desmosome and TJ organization were investigated at the mRNA and protein levels using qRT-PCR and immunohistochemistry. The distribution of junction structures was visualized using confocal microscopy. Transepidermal water loss (TEWL) functional measurements were also performed. CE intracellular structural components were similarly expressed in gland-rich (SGR and AGR) and GP areas. In contrast, significantly lower extracellular protein levels of (corneo)desmosomes (DSG1 and CDSN) and TJs (OCLN and CLDN1) were detected in SGR/AGR areas compared to GP areas. In parallel, kallikrein proteases were significantly higher in gland-rich regions. Moreover, gland-rich areas were characterized by prominently disorganized junction structures ((corneo)desmosomes and TJs) and significantly higher TEWL levels compared to GP skin, which exhibited a regular distribution of junction structures. According to our findings, the permeability barrier of our skin is not uniform. Gland-rich areas are characterized by weaker permeability barrier features compared with GP regions. These findings have important clinical relevance and may explain the preferred localization of acantholytic skin diseases on gland-rich skin regions (e.g., Pemphigus foliaceus, Darier’s disease, and Hailey–Hailey disease).

A case for the growth of ancient ooids within the sediment pile

ABSTRACT In modern ooid-forming environments in the Caribbean, aerobic respiration of organic matter below the sediment–water interface drives an increase in pCO2 and a corresponding decrease in carbonate saturation state (Ω) that creates shallow sediment porewater that is neutral or slightly caustic to carbonate. The locus of ooid growth, therefore, is presumed to be in the water column during suspension, where supersaturation with respect to calcium carbonate is the norm. In the past, however, during conditions of low aqueous O2, high Ω, or low organic-matter input, the shallow sub-sediment marine burial environment was conducive to carbonate precipitation. Here we present petrographic and electron probe microanalyzer (EPMA) data from exquisitely preserved oolites through time that suggests that some ancient ooids may have grown within the sediment pile. We propose that each increment of ooid cortical growth originated as incipient isopachous marine cement formed during shallow burial within migrating ooid dunes. After a period of burial (∼ weeks to months), ooids were remobilized and rounded during bedload transport. This “bedform model” for ooid growth explains: 1) why ancient ooids are not limited by the precipitation–abrasion balance that appears to prohibit modern tangential Caribbean ooids from achieving grain sizes larger than coarse sand, 2) the radial crystal fabric that defines the internal structure of many ancient ooids, and 3) the first-order correlation of the abundance of large and giant ooids in the rock record to periods with predicted high porewater Ω. This model implies that photosynthetic microbes were unimportant for growth of large and giant ooid but it remains agnostic to the effect of other microbes. The physical and chemical milieu of modern marine ooid-forming environments is perhaps not the best analogue for ancient ooid-forming environments; this should be considered when using ancient ooids to reconstruct secular trends in ocean chemistry.

Variant anatomy of the biliary system as a cause of pancreatic and peri-ampullary cancers

BackgroundThe cause of most pancreatic and periampullary cancers (PAC) is unknown. Recently, anatomic variations such as pancreatobiliary maljunction have been recognized as risk factors, similar to Barrett-related gastro-esophageal cancers. MethodsPre-operative MRI from 860 pancreatic/biliary resections, including 322 PACs, were evaluated for low-union (cystic duct joining the common hepatic duct inside of the pancreas or within 5 mm of the pancreatic border) ResultsLow-union, seen <10% of the population, was present in 44% of PACs (73% distal bile duct/cholangiocarcinoma, 42% pancreatic head, and 34% ampullary). It was significantly lower(11%) in conditions without connection to the ductal system (thus not exposed to the ductal/biliary tract contents), namely mucinous cystic neoplasms and intrahepatic cholangiocarcinomas(p < 0.0001). Intra-pancreatic type low-union was seen in 16% of PACs versus 2% of controls(p < 0.0001). DiscussionThis study establishes an association between low-union and PACs, and points to an anatomy-induced chemical/bilious carcinogenesis. This may explain why most pancreas cancers are in the head. It is possible that the same chemical milieu, caused by conditions other than low-union/insertion, may also play a role in the remaining half of PACs. This opens various treatment opportunities including milieu modifications (chemoprevention), focused screening of at-risk patients, and early detection with possible corrective actions.

77Se NMR Probes the Protein Environment of Selenomethionine.

Sulfur is critical for the correct structure and proper function of proteins. Yet, lacking a sensitive enough isotope, nuclear magnetic resonance (NMR) experiments are unable to deliver for sulfur in proteins the usual wealth of chemical, dynamic, and structural information. This limitation can be circumvented by substituting sulfur with selenium, which has similar physicochemical properties and minimal impact on protein structures but possesses an NMR compatible isotope (77Se). Here we exploit the sensitivity of 77Se NMR to the nucleus' chemical milieu and use selenomethionine as a probe for its proteinaceous environment. However, such selenium NMR spectra of proteins currently resist a reliable interpretation because systematic connections between variations of system variables and changes in 77Se NMR parameters are still lacking. To start narrowing this knowledge gap, we report here on a biological 77Se magnetic resonance data bank based on a systematically designed library of GB1 variants in which a single selenomethionine was introduced at different locations within the protein. We recorded the resulting isotropic 77Se chemical shifts and relaxation times for six GB1 variants by solution-state 77Se NMR. For four of the GB1 variants we were also able to determine the chemical shift anisotropy tensor of SeM by solid-state 77Se NMR. To enable interpretation of the NMR data, the structures of five of the GB1 variants were solved by X-ray crystallography to a resolution of 1.2 Å, allowing us to unambiguously determine the conformation of the selenomethionine. Finally, we combine our solution- and solid-state NMR data with the structural information to arrive at general insights regarding the execution and interpretation of 77Se NMR experiments that exploit selenomethionine to probe proteins.

243 Permeability and immune barrier differences of healthy human skin

Despite the distinct microbial and chemical milieu on topographically distinct skin areas, the immunological and permeability barrier of the healthy skin has been previously considered to be unified on the whole body surface. We provided the first evidence that sebaceous gland rich (SGR) and sebaceous gland poor (SGP) regions exhibit a characteristic innate and adaptive immune milieu (increased chemokine expression and a homeostatic IL-17 dominance in SGR skin). As a next step, we aimed to investigate at the molecular level whether permeability barrier of these regions are also different. We analyzed the expression of the most important barrier structure (FLG, KRT1, 6A, 10, 16, 17, 79, LCE1D, 1F, LOR, SPRR1A, 2A), tight junction and desmosome components (CDSN, CLDN1, 16, 23, CDH1, DSC1, DSG1, OCLN), enzymes related to barrier formation (TGM1, 3, 5, KLK5, 7, 14) and antimicrobial peptides (AMPs; S100A7,8,9, LCN2, hBD2) both at the mRNA and protein levels by RT-qPCR and IHC. Regarding barrier structure molecules and junction components KRT17 and 79 were found to be significantly differentially expressed with higher levels in SGR skin whereas amongst the examined enzymes KLK5 and 7 showed significantly higher levels in SGR at the gene level. Moreover, all investigated AMPs were significantly upregulated in SGR. At the protein level, KRT1, KRT17, S100A8 and LCN2 showed significantly elevated level in SGR skin among the investigated molecules. Similarly to the prominently different immune tuning of SGP and SGR region we could also detect barrier differences between the distinct skin regions which together may explain the characteristic localization of certain immune mediated skin disorders and distinct skin sites may require distinct barrier repair therapies.

Rational design of gelatin/nanohydroxyapatite cryogel scaffolds for bone regeneration by introducing chemical and physical cues to enhance osteogenesis of bone marrow mesenchymal stem cells.

Identification of key components in the chemical and physical milieu for directing osteogenesis is a requirement in the investigation of tissue engineering scaffolds for advancement of bone regeneration. In this study, we engineered different gelatin-based cryogels and studied the effect of nanohydroxyapatite (nHAP) and crosslinking agents on scaffold properties and its osteogenic response towards bone marrow stem cells (BMSCs). The cryogels examined are 5% gelatin and 5% gelatin/2.5% nHAP, crosslinked either with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or glutaraldehyde (GA). We confirmed that nHAP or the crosslinking agent has no effects on scaffold pore size and porosity. Nonetheless, incorporation of nHAP increased mechanical strength, swelling ratio and degree of crosslinking, but decreased degradation rate. Cryogels crosslinked with EDC showed faster degradation and promoted osteogenic differentiation of BMSCs while those prepared from GA crosslinking promoted proliferation of BMSCs. Furthermore, osteogenic differentiation was always enhanced in the presence of nHAP irrespective of the culture medium (normal or osteogenic) used but osteogenic medium always provide a higher extent of osteogenic differentiation. Employing gelatin/nHAP cryogel crosslinked by EDC in a bioreactor for dynamic culture of BMSCs, cyclic compressive mechanical simulation was found to be beneficial for both cell proliferation and osteogenic differentiation. However, the optimum conditions for osteogenic differentiation and cell proliferation were found at 30% and 60% strain, respectively. We thus demonstrated that osteogenic differentiation of BMSCs could be tuned by taking advantages of chemical cues generated from scaffold chemistry or physical cues generated from dynamic cell culture in vitro. Furthermore, by combining the best cryogel preparation and in vitro cell culture condition for osteogenesis, we successfully employed in vitro cultured cryogel/BMSCs constructs for repair of rabbit critical-sized cranial bone defects.

Extending Arms of Insulin Resistance from Diabetes to Alzheimer's Disease: Identification of Potential Therapeutic Targets.

Type 3 diabetes (T3D) is chronic insulin resistant state of brain which shares pathology with sporadic Alzheimer's disease (sAD). Insulin signaling is a highly conserved pathway in the living systems that orchestrate cell growth, repair, maintenance, energy homeostasis and reproduction. Although insulin is primarily studied as a key molecule in diabetes mellitus, its role has recently been implicated in the development of Alzheimer's disease (AD). Severe complications in brain of diabetic patients and metabolically compromised status is evident in brain of AD patients. Underlying shared pathology of two disorders draws a trajectory from peripheral insulin resistance to insulin unresponsiveness in the central nervous system (CNS). As insulin has a pivotal role in AD, it is not an overreach to address diabetic condition in AD brain as T3D. Insulin signaling is indispensable to nervous system and it is vital for neuronal growth, repair, and maintenance of chemical milieu at synapses. Downstream mediators of insulin signaling pathway work as a regulatory hub for aggregation and clearance of unfolded proteins like Aβ and tau. In this review, we discuss the regulatory roles of insulin as a pivotal molecule in brain with the understanding of defective insulin signaling as a key pathological mechanism in sAD. This article also highlights ongoing trials of targeting insulin signaling as a therapeutic manifestation to treat diabetic condition in brain.