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Obesity, chronic pain, and aging are prevalent global challenges with profound implications for health and well-being. Central to these processes are adrenal hormones, particularly cortisol and dehydroepiandrosterone (DHEA), along with its sulfated form (DHEAS). Cortisol, essential for stress adaptation, can have adverse effects on pain perception and aging when dysregulated, while DHEA/S possess properties that may mitigate these effects. This review explores the roles of cortisol and DHEA/S in the contexts of obesity, acute and chronic pain, aging, and age-related diseases. We examine the hormonal balance, specifically the cortisol-to-DHEA ratio (CDR), as a key marker of stress system functionality and its impact on pain sensitivity, neurodegeneration, and physical decline. Elevated CDR and decreased DHEA/S levels are associated with worsened outcomes, including increased frailty, immune dysfunction, and the progression of age-related conditions such as osteoporosis and Alzheimer's disease. This review synthesizes the current literature to highlight the complex interplay between these hormones and their broader implications for health. It aims to provide insights into potential future therapies to improve pain management and promote healthy weight and aging. By investigating these mechanisms, this work contributes to a deeper understanding of the physiological intersections between pain, aging, and the endocrine system.

A comprehensive solution of a laser diode (LD) driver with temperature control using a programmable system on a chip is proposed as an alternative to dedicated devices. A digital proportional integral differential controller is used for regulating the LD temperature. The light-current characteristics and frequency response of the LD are measured using an external photodiode and interrogated by an integrated transimpedance amplifier. The 0.084% stability, 2 kHz bandwidth, and 0.11% full scale current error are demonstrated. The LD overshoot protection is digitally implemented, providing a soft start. While the circuit is initially optimized for a 3 W infrared LD, instructions are provided for fine-tuning the design according to specific LD requirements., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Multidetector computed tomography (MDCT) is often necessary to manufacture 3D-printed medical models (MMs) required for mandibular restoration due to trauma or malignant tumor. Although cone-beam computed tomography (CBCT) is a preferable method of mandibular imaging, additional scanning is often unjustified. To test whether a single radiologic protocol could be used for mandibular reconstructions, the human mandible was scanned with 6 MDCT and 2 CBCT protocols and later 3D-printed using a fused-deposition modelling technique. Then, we assessed linear measures on the mandible and compared them with MDCT/CBCT digital scans and 3D-printed MMs. Our data revealed that CBCT0.25 was the most precise protocol for manufacturing 3D-printed mandibular MMs, which is expected considering its voxel size. However, we noted that CBCT0.35 and Dental2.0H60s MDCT protocols were of comparable accuracy, indicating that this MDCT protocol could be a single radiologic protocol used to scan both donor and recipient regions required for mandibular reconstruction., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 by Mutaz B. Habal, MD.)

Scapula tip flaps have been introduced in the literature as an ideal surgical treatment option for large defects in the horizontal plane of the maxilla. This article aims to present a unique step by step protocol for a near total maxillectomy with a pterygoid bone resection and consecutive microvascular reconstruction with a harvested scapula tip flap. The protocol includes immediate placement of extra-short implants in donor bone with the aid of Virtual Surgical Planning (VSP), and an in-house 3D printing of medical 3D models and surgical guides. So far, there has been no presented surgical technique combining immediate implant placement in the scapula region with simultaneous microvascular repair. This technique allows: tumour resection; flap harvesting; extra-short implant placements and reconstruction to be performed in one simultaneous procedure. The technique is presented with illustrations, VSP (presented on videos), radiographs, and surgical findings. We discovered that this refinement of the scapula tip surgery has enabled reconstructive procedures to be performed at the same time as implant placements, providing expedited functional and aesthetic outcomes in selected cases. Moreover, modification of the surgical technique could enhance the competence of the oropharyngeal edge. In conclusion, this new surgical protocol utilizing VSP, 3D models and simultaneous extra-short implant placement provides indispensable advantages for such a complicated surgical procedures, while significantly shortening the duration of surgery., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Background: The purpose of this study was to assess the clinical presentation and contemporary management of arterial thoracic outlet syndrome (TOS) in high-volume referral center., Methods: We conducted a retrospective review of a prospectively maintained database of patients with TOS of any etiology between January 1 st 1990 and 2021. Supra-, or combined supra-/infraclavicular approaches have been used for decompression/vascular reconstructions. The group was divided into two equal time periods: period 1 (1990-2006, N.=27) and period 2 (2006-2021, N.=36)., Results: Sixty-three consecutive patients underwent surgical treatment due to arterial TOS. Period 2 had more patients who were asymptomatic (N.=16, 44.4% vs. N.=0, 0%, P<0.001) and those presenting with critical hand ischemia (N.=12, 33.3% vs. N.=0, 0%, P=0.01), while acute limb ischemia was more common in period 1 (N.=16, 59.2% vs. N.=5, 13.9%, P<0.001). SA compression without lesion was more common in period 2 (N.=16, 44.4% vs. N.=0, 0%, P<0.001), while SA intimal damage with mural thrombus formation was more common in the period 1 (N.=12, 44.4% vs. N.=1, 2.7%, P<0.001). Decompression as an isolated procedure was performed in 25.4% (N.=16) of all asymptomatic patients, while combined decompressive and vascular procedure in 71.4% (N.=45) of patients. The most common postoperative complication was pneumothorax (N.=7, 11.1%)., Conclusions: The supraclavicular approach with its modifications provides adequate decompression and allows also repair or reconstruction of the SA, as well as complete additional revascularization of the upper extremity without the need for further patient repositioning. While treatment methods and early outcomes have not changed significantly over time, there has been a trend towards different clinical and SA pathomorphological presentation.

This study investigates the interfacial behavior of the proteinase K enzyme at air-water interface. Adsorption of enzyme on the surface was induced using saline subphase. The surface packing and stability of the enzyme was investigated using of surface pressure-area (π-A) and surface potential-area (ΔV-A) isotherms. Proteinase K enzyme forms film at air-aqueous interface and demonstrates good stability as shown through compression-decompression cycle experiments. To characterize the surface assembly morphology of the interfacial enzymes UV-vis and fluorescence spectroscopic techniques were used. The data revealed that the enzyme Langmuir monolayer has good homogeneity with no evidence of aggregates during compression. The secondary structure of the enzyme at interface was determined to be α-helix using p-polarized infrared-reflection absorption spectroscopy. This was confirmed through Circular dichroism spectra of the enzyme Langmuir-Blodgett (LB) film which showed that the major conformation present were α-helices., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The aim of this study was to develop novel hydroxyapatite (HAP)-based bioactive bone replacement materials for segmental osteotomy reconstruction. Customized three-dimensional (3D) bone construct was manufactured from nanohydroxyapatite (nHAP) with poly(lactide-co-glycolide) (PLGA) coating using 3D models derived from the computed tomography (CT) scanning of the rabbit's ulna and gradient 3D printing of the bone substitute mimicking the anatomical shape of the natural bone defect. Engineered construct revealed adequate micro-architectural design for successful bone regeneration having a total porosity of 64% and an average pore size of 256 μm. Radiography and micro-CT analysis depicted new bone apposition through the whole length of the reconstructed ulna with a small area of non-resorbed construct in the central area of defect. Histological analysis revealed new bone formation with both endochondral and endesmal type of ossification. Immunohistochemistry analysis depicted the presence of bone formation indicators - bone morphogenetic protein (BMP), osteocalcin (OCN) and osteopontin (OPN) within newly formed bone. Manufactured personalized construct acts as a "smart" responsive biomaterial capable of modulating the functionality and potential for the personalized bone reconstruction on a clinically relevant length scale.

Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of κM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJ's exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics.

Nanoparticles have been conjugated to biological systems for numerous applications such as self-assembly, sensing, imaging, and therapy. Development of more reliable and robust biosensors that exhibit high response rate, increased detection limit, and enhanced useful lifetime is in high demand. We have developed a sensing platform by the conjugation of β-galactosidase, a crucial enzyme, with lab-synthesized gel-like carbon dots (CDs) which have high luminescence, photostability, and easy surface functionalization. We found that the conjugated enzyme exhibited higher stability towards temperature and pH changes in comparison to the native enzyme. This enriched property of the enzyme was distinctly used to develop a stable, reliable, robust biosensor. The detection limit of the biosensor was found to be 2.9 × 10 -4 M, whereas its sensitivity was 0.81 µA·mmol -1 ·cm -2 . Further, we used the Langmuir monolayer technique to understand the surface properties of the conjugated enzyme. It was found that the conjugate was highly stable at the air/subphase interface which additionally reinforces the suitability of the use of the conjugated enzyme for the biosensing applications.

In this article, we explored the surface chemistry properties of a cholera toxin B (CTB) monolayer at the air-subphase interface and investigated the change in interfacial properties through in situ spectroscopy. The study showed that the impact of the blue shift was negligible, suggesting that the CTB molecules were minimally affected by the subphase molecules. The stability of the CTB monolayer was studied by maintaining the constant surface pressure for a long time and also by using the compression-decompression cycle experiments. The high stability of the Langmuir monolayer of CTB clearly showed that the driving force of CTB going to the amphiphilic membrane was its amphiphilic nature. In addition, no major change was detected in the various in situ spectroscopy results (such as UV-vis, fluorescence, and IR ER) of the CTB Langmuir monolayer with the increase in surface pressure. This indicates that no aggregation occurs in the Langmuir monolayer of CTB.

This article investigates the surface chemistry properties of the β-galactosidase monolayer at the air-subphase interface at the vicinity of its substrate, X-gal. We have demonstrated that the β-galactosidase in the monolayer form remained active and performed hydrolysis of the X-gal in the subphase. We investigated the β-galactosidase Langmuir monolayer in absence and presence of X-gal in the subphase of varying concentration of X-gal with the sodium chloride solution. It was found that the limiting molecular area as well as the collapse surface pressure kept on decreasing with the increasing concentration of X-gal. In accordance to the data obtained from the isotherm it was also found that β-galactosidase forms a stable monolayer that does not aggregate at the air-subphase interface. The stability of the monolayer at the air-subphase interface was studied by using compression-decompression cycles with and without X-gal at varying concentration and different surface pressures. The infrared reflection-absorption spectroscopy (IRRAS) and Brewster angle microscopy (BAM) of β-galactosidase Langmuir monolayer was also investigated for pure and mixed β-galactosidase at the air-subphase.

The changes of interfacial properties of β-galactosidase introduced into different pH environments are investigated through surface chemistry and in situ spectroscopy. Conditions for an optimal Langmuir monolayer formation were firstly obtained by varying the subphase salt concentration and the surface-pressure area isotherm was used to extrapolate the limiting molecular area of the enzyme monolayer to be around 42,000 Å(2) molecule(-1). Surface pressure stability measurements held at 20 mN/m for 90 min along with compression-decompression cycles revealed no aggregate formation at the air-water interface. Consistent with the data obtained from the isotherm, in situ UV-Vis and fluorescence spectroscopy shows a steep rise in absorbance and photoluminescence intensity correlating to with a switch from a liquid-expanded to a liquid-condensed phase. A decrease in subphase pH increased the electrostatic repulsion as the enzyme was protonated, leading to an expanded monolayer. Infrared absorption-reflection spectroscopy demonstrates that the enzyme adopts mainly β-sheet conformation at the air-water interface before and during the compression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Biosensing methods and devices using graphene oxide (GO) have recently been explored for detection and quantification of specific biomolecules from body fluid samples, such as saliva, milk, urine, and serum. For a practical diagnostics application, any sensing system must show an absence of nonselective detection of abundant proteins in the fluid matrix. Because lysozyme is an abundant protein in these body fluids (e.g., around 21.4 and 7 μg/mL of lysozyme is found in human milk and saliva from healthy individuals, and more than 15 or even 100 μg/mL in patients suffering from leukemia, renal disease, and sarcoidosis), it may interfere with detections and quantification if it has strong interaction with GO. Therefore, one fundamental question that needs to be addressed before any development of GO based diagnostics method is how GO interacts with lysozyme. In this study, GO has demonstrated a strong interaction with lysozyme. This interaction is so strong that we are able to subsequently eliminate and separate lysozyme from aqueous solution onto the surface of GO. Furthermore, the strong electrostatic interaction also renders the selective adsorption of lysozyme on GO from a mixture of binary and ternary proteins. This selectivity is confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), fluorescence spectroscopy, and UV-vis absorption spectroscopy.

Fluorescent insulin fibrils gold nanoclusters (Au NCs) have been synthesized through the reduction of gold by human insulin in fibrillated form. Likewise, nanocluster formation has been regulated by insulin, working as a protein-based template. Environment- and surface-controlled experiments have shown the optimized synthesis conditions is comprised of a pure aqueous alkaline solvent for insulin under constant heat at physiological temperature (37°C) prior to addition of the Au precursor (HAuCl4), followed by subsequent heating (37°C) and vigorous stirring after the addition of HAuCl4 until the completion of the synthetic approach. Microscopy experiments detected the presence of primordial fibril structures in samples of heated human insulin in the alkaline medium prior to addition of HAuCl4, while encountering more developed insulin fibrils in the terminal production of Au NCs. This investigation provides insight to the development of a novel synthesis of Au NCs in the alkaline medium, while providing a graphical description of the environmental and surface-dependent effects that were presented in the synthesis of human insulin nanoclusters. The study provides pertinent information for future synthetic procedures, as the protein state of several protein-nanoparticle systems may reflect on the results that were obtained herein., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Human islet amyloid polypeptide (hIAPP) is the source of the major component of the amyloid deposits found in the islets of Langerhans of around 95 per cent type 2 diabetic patients. The formation of aggregates and mature fibrils is thought to be responsible for the dysfunction and death of the insulin-producing pancreatic β-cells. Investigation on the conformation, orientation and self-assembly of the hIAPP at time zero could be beneficial for our understanding of its stability and aggregation process. To obtain these insights, the hIAPP at time zero was studied at the air-aqueous interface using the Langmuir monolayer technique. The properties of the hIAPP Langmuir monolayer at the air-aqueous interface on a NaCl subphase with pH 2.0, 5.6 and 9.0 were examined by surface pressure- and potential-area isotherms, UV-Vis absorption, fluorescence spectroscopy and Brewster angle microscopy. The conformational and orientational changes of the hIAPP Langmuir monolayer under different surface pressures were characterized by p-polarized infrared-reflection absorption spectroscopy, and the results did not show any prominent changes of conformation or orientation. The predominant secondary structure of the hIAPP at the air-aqueous interface was α-helix conformation, with a parallel orientation to the interface during compression. These results showed that the hIAPP Langmuir monolayer at the air-aqueous interface was stable, and no aggregate or domain of the hIAPP at the air-aqueous interface was observed during the time of experiments.

The human insulin (HI) protein was examined to elucidate its structure at the air-water interface. Optimal experimental conditions were determined to prepare a homogeneous and stable human insulin (HI) Langmuir monolayer. HI insulin Langmuir monolayer can be used to study interactions of HI with a membrane as Langmuir monolayers are used as an in vitro model of biological membranes. Surface pressure and surface potential-area isotherms were used to characterize the HI Langmuir monolayer. The compression-decompression cycles and stability measurements showed a homogeneous and stable monolayer at the air-water interface. However, higher surface pressures resulted in a higher decrease in area and less stability. In situ UV-vis and fluorescence spectroscopy were used to verify the homogeneity of the HI monolayer and to identify the chromophore residues in the HI. Domain formation was examined through epifluorescence and Brewster angle microscopies. The conformation of HI was examined by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) in the aqueous phase and at the air-water interface by infrared reflection absorption spectroscopy (IRRAS). HI was found to exist as a monomer in 2-D.

The human insulin (HI) Langmuir monolayer at the air-water interface was systematically investigated in the presence and absence of Zn(II) ions in the subphase. HI samples were dissolved in acidic (pH 2) and basic (pH 9) aqueous solutions and then spread at the air-water interface. Spectroscopic data of aqueous solutions of HI show a difference in HI conformation at different pH values. Moreover, the dynamics of the insulin protein showed a dependence on the concentration of Zn(II) ions. In the absence of Zn(II) ions in the subphase, the acidic and basic solutions showed similar behavior at the air-water interface. In the presence of Zn(II) ions in the subphase, the surface pressure-area and surface potential-area isotherms suggest that HI may aggregate at the air-water interface. It was observed that increasing the concentration of Zn(II) ions in the acidic (pH 2) aqueous solution of HI led to an increase of the area at a specific surface pressure. It was also seen that the conformation of HI in the basic (pH 9) medium had a reverse effect (decrease in the surface area) with the increase of the concentration of Zn(II) ions in solution. From the compression-decompression cycles we can conclude that the aggregated HI film at air-water interface is not stable and tends to restore a monolayer of monomers. These results were confirmed from UV-vis and fluorescence spectroscopy analysis. Infrared reflection-absorption and circular dichroism spectroscopy techniques were used to determine the secondary structure and orientation changes of HI by zinc ions. Generally, the aggregation process leads to a conformation change from α-helix to β-strand and β-turn, and at the air-water interface, the aggregation process was likewise seen to induce specific orientations for HI in the acidic and basic media. A proposed surface orientation model is presented here as an explanation to the experimental data, shedding light for further research on the behavior of insulin as a Langmuir monolayer.

Lipid rafts being rich in cholesterol and sphingolipids are considered to provide ordered lipid environment in the neuronal membranes, where it is hypothesized that the cleavage of amyloid precursor protein (APP) to Aβ (1-40) and Aβ (1-42) takes place. It is highly likely that the interaction of lipid raft components like cholesterol, sphingomylein or GM1 leads to nucleation of Aβ and results in aggregation or accumulation of amyloid plaques. One has investigated surface pressure-area isotherms of the lipid raft and Aβ (1-40) Langmuir monolayer. The compression-decompression cycles and the stability of the lipid raft Langmuir monolayer are crucial parameters for the investigation of interaction of Aβ (1-40) with the lipid raft Langmuir monolayer. It was revealed that GM1 provides instability to the lipid raft Langmuir monolayer. Adsorption of Aβ (1-40) onto the lipid raft Langmuir monolayer containing neutral (POPC) or negatively charged phospholipid (DPPG) was examined. The adsorption isotherms revealed that the concentration of cholesterol was important for adsorption of Aβ (1-40) onto the lipid raft Langmuir monolayer containing POPC whereas for the lipid raft Langmuir monolayer containing DPPG:cholesterol or GM1 did not play any role. In situ UV-vis absorption spectroscopy supported the interpretation of results for the adsorption isotherms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A novel, centrifugal disk-based micro-total analysis system (μTAS) for low cost and high throughput semi-automated immunoassay processing was developed. A key innovation in the disposable immunoassay disk design is in a fluidic structure that enables very efficient micro-mixing based on a reciprocating mechanism in which centrifugal acceleration acting upon a liquid element first generates and stores pneumatic energy that is then released by a reduction of the centrifugal acceleration, resulting in a reversal of direction of flow of the liquid. Through an alternating sequence of high and low centrifugal acceleration, the system reciprocates the flow of liquid within the disk to maximize incubation/hybridization efficiency between antibodies and antigen macromolecules during the incubation/hybridization stage of the assay. The described reciprocating mechanism results in a reduction in processing time and reagent consumption by one order of magnitude., (© 2011 American Institute of Physics)

Nanoparticles have enormous potential in diagnostic and therapeutic studies. We have demonstrated that the amyloid beta mixed with and conjugated to dihydrolipoic acid- (DHLA) capped CdSe/ZnS quantum dots (QDs) of size approximately 2.5 nm can be used to reduce the fibrillation process. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used as tools for analysis of fibrillation. There is a significant change in morphology of fibrils when amyloid β (1-42) (Aβ (1-42)) is mixed or conjugated to the QDs. The length and the width of the fibrils vary under modified conditions. Thioflavin T (ThT) fluorescence supports the decrease in fibril formation in presence of DHLA-capped QDs.

Human cardiac troponin I (cTnI) is the preferred biomarker in the assessment of myocardial infarction. It is known to interact with troponin C and T to form a trimeric complex. Whereas small amounts are found in the cytoplasm, most of cTnI is in the form of a complex with actin located in myofilaments. To understand these interactions of cTnI better, we first investigated the surface chemistry of cTnI as a Langmuir monolayer spread at the air-water interface. We investigated the optimal conditions for obtaining a stable Langmuir monolayer in terms of changing the ionic strength of the subphase using different concentrations of potassium chloride. Monolayer stability was investigated by compressing the cTnI monolayer to a specific surface pressure and keeping the surface pressure constant while measuring the decrease in the molecular area as a function of time. Aggregation and/or domain formation was investigated by using compression-decompression cycles, in situ UV-vis spectroscopy, Brewster angle microscopy (BAM), and epifluorescence microscopy. To ensure that the secondary structure is maintained, we used infrared reflection-absorption spectroscopy (IRRAS) directly at the air-subphase interface. It was found that cTnI forms a very stable monolayer (after more that 5000 s) that does not aggregate at the air-subphase interface. The cTnI molecules maintain their secondary structure and, on the basis of the low reflectivity observed using BAM measurements and the low reflection-absorption intensities measured with IRRAS spectroscopy, lie flat on the subphase with the alpha-helices parallel to the air-subphase interface.

Amyloid beta (1-40) and (1-42) peptides are the major constituents of hallmark senile plaques found in Alzheimer's disease (AD) patients. Study of aggregation of Abeta (1-40) and (1-42) peptides and the truncated Abeta fragments could lead towards the mechanism of AD. Langmuir monolayer approach is one of the excellent methods to investigate the mechanism and origin of AD. Particularly, to study the steps involved in the formation and assembly of beta-sheet structures leading to formation of amyloid fibrils. Surface pressure- and surface potential-area isotherms provide information regarding the nature of short-range and long-range interactions between the molecules especially the lipids and the Abeta peptides. Spectroscopic methods like IRRAS, PM-IRRAS, FTIR-ATR, and GIXD at the air-water interface provide insight into the structural characterization, and orientation of the molecules in the Langmuir monolayer.

Proper mixing of reagents is of paramount importance for an efficient chemical reaction. While on a large scale there are many good solutions for quantitative mixing of reagents, as of today, efficient and inexpensive fluid mixing in the nanoliter and microliter volume range is still a challenge. Complete, i.e., quantitative mixing is of special importance in any small-scale analytical application because the scarcity of analytes and the low volume of the reagents demand efficient utilization of all available reaction components. In this paper we demonstrate the design and fabrication of a novel centrifugal force-based unit for fast mixing of fluids in the nanoliter to microliter volume range. The device consists of a number of chambers (including two loading chambers, one pressure chamber, and one mixing chamber) that are connected through a network of microchannels, and is made by bonding a slab of polydimethylsiloxane (PDMS) to a glass slide. The PDMS slab was cast using a SU-8 master mold fabricated by a two-level photolithography process. This microfluidic mixer exploits centrifugal force and pneumatic pressure to reciprocate the flow of fluid samples in order to minimize the amount of sample and the time of mixing. The process of mixing was monitored by utilizing the planar laser induced fluorescence (PLIF) technique. A time series of high resolution images of the mixing chamber were analyzed for the spatial distribution of light intensities as the two fluids (suspension of red fluorescent particles and water) mixed. Histograms of the fluorescent emissions within the mixing chamber during different stages of the mixing process were created to quantify the level of mixing of the mixing fluids. The results suggest that quantitative mixing was achieved in less than 3 min. This device can be employed as a stand alone mixing unit or may be integrated into a disk-based microfluidic system where, in addition to mixing, several other sample preparation steps may be included.

Alzheimer disease (AD) patients have an impairment of anti-amyloid-beta (Abeta) innate immunity and a defect in immune gene transcription [Fiala, M., Liu, P.T., Espinosa-Jeffrey, A., Rosenthal, M.J., Bernard, G., Ringman, J.M., Sayre, J., Zhang, L., Zaghi, J., Dejbakhsh, S., Chiang, B., Hui, J., Mahanian, M., Baghaee, A., Hong, P., Cashman, J., 2007b. Innate immunity and transcription of MGAT-III and Toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin. Proc. Natl. Acad. Sci. U. S. A. 104, 12849-12854]. Early diagnosis is a cornerstone of preventive approaches to AD. Phospho-tau and Abeta CSF levels are useful markers of neurodegeneration but not of a process leading to neurodegeneration. To detect an early biomarker of AD, we developed a flow cytometric test of Abeta phagocytosis, which was 94% positive (<400 MFI units) in AD patients (mean age+/-SEM 77+2.2 years; mean score+/-SEM 198.6+/-25.5 MFI units) and 60% positive in MCI patients (77+/-5.6 years; 301+/-106 MFI units). Control subjects, active senior university professors, were 100% negative (74.2+/-4.2 years; 1348+/-174 MFI units). The test had a low specificity in older caregivers and older amyotrophic lateral sclerosis (ALS) patients. We also tested transcriptional regulation of the genes MGAT-III and Toll-like receptor-3 in macrophages. Macrophages of "Type I" patients (a majority of patients) showed gene down regulation at baseline and up regulation by curcuminoids; macrophages of "Type II" patients showed opposite responses. The results of flow cytometric testing suggest that normal Abeta phagocytosis is associated with healthy cognition and lesser risk of AD. The significance of abnormal results in aged persons should be investigated by prospective studies to determine the risk of AD.

Problem: Studies on experimental antiphospholipid syndrome (APS) models proved that molecular mimicry between plasma protein beta(2)-glycoprotein I (beta(2)GPI) and structure within micro-organisms or their products, might be a cause for experimental APS. Considering the heterogeneity of polyclonal antiphospholipid antibodies (aPLs), it is important to define the precise characteristics of pathogenic aPLs. To avoid the influence of polyclonality and to further analyse the connection between molecular mimicry and APS, we produced monoclonal antibodies (MAbs) against tetanus toxoid (TTd) and tested their reactivity against beta(2)GPI., Method of Study: In this report, we analysed the characteristics of MAb26 raised against TTd and cross-reactive with beta(2)GPI: its binding properties in various in vitro immunoassays, its specific interactions with surface epitopes expressed on apoptotic cells and its role in vivo., Results: We have demonstrated that MAb26: (i) binds beta(2)GPI being immobilized on an appropriate surface: irradiated polystyrene plates, non-irradiated plates pre-coated with anionic phospholipids and polyvinylidene fluoride membrane; (ii) binds specifically to apoptotic but not to viable cells and the binding is beta(2)GPI-dependent; and (iii) induces a pathologic pregnancy outcome when passively injected into BALB/c mice., Conclusion: This study concluded that certain subpopulations of antibodies raised against TTd and cross-reactive with beta(2)GPI, because of the molecular mimicry mechanism, could have pathologic potential.

Gold quantum dots (AuQDs) were synthesized and electrostatically conjugated to goat-derived anti-human IgG for the purpose of detecting human IgG in solution over a broad range of concentrations. The system is able to detect human IgG by linear fluorescence quenching over a micromolar to nanomolar concentration range. We have demonstrated the specificity and a wide dynamic range of the proposed immunoassay. The quenching is a result of competitive surface quenching of the AuQDs. Characterization, details of the immunoassay, and the quenching mechanism, are discussed.

The Langmuir monolayer of aequorin and apoaequorin was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS techniques. The alpha-helices in the aequorin Langmuir monolayer were parallel to the air-water interface at zero surface pressure. When the surface pressure increased to 15 mN.(m-1), the alpha-helices became tilted and the turns became parallel to the air-water interface. As for apoaequorin, the alpha-helices were also parallel to the air-water interface at 0 mN.m(-1). However, the alpha-helix became tilted and the turns became parallel to the air-water interface quickly at 5 mN.m(-1). With further compression of the apoaequorin Langmuir monolayer, the orientation remained the same. The different behaviors of aequorin and apoaequorin at the air-water interface were explained by the fact that aequorin formed dimers at the air-water interface but apoaequorin was a monomer. It is more difficult for a dimer to be tilted by the compression of the Langmuir monolayer.

In this paper, we studied the surface properties of recombinant aequorin at the air-water interface. Using the Langmuir monolayer technique, the surface properties of aequorin were studied, including the surface pressure and surface potential-area isotherms, compression-decompression cycles, and stability on Trizma Base (Tris/HCl) buffer at pH 7.6. The results showed that aequorin formed a stable Langmuir monolayer and the surface pressure-area isotherms were dependent on both pH and ionic strength. At a pH higher or lower than 7.6, the limiting molecular area decreased. The circular dichroism (CD) spectra of aequorin in aqueous solutions explained this result: when the pH was higher than 7.6, the alpha-helix conformation changed to unordered structures, whereas at a pH lower than 7.6, the alpha-helix conformation changed to beta-sheet. The addition of calcium chloride to the Tris/HCl buffer subphase (pH 7.6) caused an increase of the limiting molecular area of the aequorin Langmuir monolayer. The fluorescence spectra of a Langmuir-Blodgett (LB) film of aequorin in the presence of calcium chloride indicated that the aequorin transformed to the apoaequorin.

In this paper, we present the design and characterization of a novel platform for mechanical cell lysis of even the most difficult to lyse cell types on a micro or nanoscale (maximum 70 microL total volume). The system incorporates a machined plastic circular disk assembly, magnetic field actuated microfluidics, centrifugal cells and tissue homogenizer and centrifugation system. The mechanism of tissue disruption of this novel cell homogenization apparatus derives from the relative motion of ferromagnetic metal disks and grinding matrices in a liquid medium within individual chambers of the disk in the presence of an oscillating magnetic field. The oscillation of the ferromagnetic disks or blades produces mechanical impaction and shear forces capable of disrupting cells within the chamber both by direct action of the blade and by the motion of the surrounding lysis matrix, and by motion induced vortexing of buffer fluid. Glass beads or other grinding media are integrated into each lysis chamber within the disk to enhance the transfer of energy from the oscillating metal blade to the cells. The system also achieves the centrifugal elimination of solids from each liquid sample and allows the elution of clarified supernatants via siphoning into a collection chamber fabricated into the plastic disk assembly. This article describes system design, implementation and validation of proof of concept on two samples--Escherichia coli and Saccharomyces cerevisiae representing model systems for cells that are easy and difficult to lyse, respectively.

An immunoassay based upon photoluminescent gold quantum dots aimed at detecting human IgG in aqueous solution from micromolar to nanomolar concentrations is described.

Gelatin having p-nitrocinnamate pendant groups (Gel-NC) was prepared via an efficient one-pot synthesis, yield >87%. (1)H NMR data indicated that 1 mol of gelatin was modified with 18 +/- 6 mol of the photosensitive group. Upon exposure to low-intensity 365 nm UV light and in the absence of photoinitiators or catalysts, Gel-NC cross-linked within minutes into a gelatin-based hydrogel as monitored by UV-vis spectroscopy. The degree of swelling of this biodegradable hydrogel in aqueous solutions responded to changes in Gel-NC concentration levels, the ionic strength of the aqueous solutions, and photo-cross-linking time. Topography changes associated with phase transition resulting from "photocleavage" of the hydrogel network with 254 nm UV light were studied with AFM. Both Gel-NC and its hydrogel expressed low toxicity to human neonatal fibroblast cells. In addition, gelatin-based microgels were prepared via the photo-cross-linking of Gel-NC within inverse micelles.

We report on single-molecule studies of photosensitized interfacial electron transfer (ET) processes in Coumarin 343 (C343)-TiO(2) nanoparticles (NP) and Cresyl Violet (CV(+))-TiO(2) NP systems, using time-correlated single-photon counting coupled with scanning confocal fluorescence microscopy. Fluorescence intensity trajectories of individual dye molecules adsorbed on a semiconductor NP surface showed fluorescence fluctuations and blinking, with time constants distributed from milliseconds to seconds. The fluorescence fluctuation dynamics were found to be inhomogeneous from molecule to molecule and from time to time, showing significant static and dynamic disorders in the interfacial ET reaction dynamics. We attribute fluorescence fluctuations to the interfacial ET reaction rate fluctuations, associating redox reactivity intermittency with the fluctuations of molecule-TiO(2) electronic and Franck-Condon coupling. Intermittent interfacial ET dynamics of individual molecules could be characteristic of a surface chemical reaction strongly involved with and regulated by molecule-surface interactions. The intermittent interfacial reaction dynamics that likely occur among single molecules in other interfacial and surface chemical processes can typically be observed by single-molecule studies but not by conventional ensemble-averaged experiments.

We report on imaging living bacterial cells by using a correlated tapping-mode atomic force microscopy (AFM) and confocal fluorescence lifetime imaging microscopy (FLIM). For optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells, we explored different methods of bacterial sample preparation, such as spreading the cells on poly-L-lysine coated surfaces or agarose gel coated surfaces. We have found that the agarose gel containing 99% ammonium acetate buffer can provide sufficient local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and signal-to-noise ratio of the AFM images. Near-field AFM-tip-enhanced fluorescence lifetime imaging (AFM-FLIM) holds high promise on obtaining fluorescence images beyond optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging bacterial living cells, we demonstrated a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging of living bacterial cells to characterize cell polarity.

In this paper, we present a nanoscale study of the supramolecular structure of the dehydrogenate polymer (ZL-DHP) lignin model compound. The combination of near-field scanning optical microscopy (NSOM or SNOM) and atomic force microscopy (AFM) has been utilized to explore physicochemical properties of the lignin model compound on a scale ranging from individual macromolecules to globular supramolecular assemblies. By utilizing NSOM in transmission mode, the optical inhomogeneity in the lignin supramolecular structure has been observed for the first time. In particular, the transmission-mode NSOM images reveal a combination of hollow and layered supramolecular globular structure in the lignin model compound. Through the paired use of TappingMode and pulsed-mode AFM, we have also confirmed the existence of regions with different rheological properties on the single lignin model compound supramolecular assembly.

In this paper we present our surface chemistry studies of enzymatically polymerized, poly-coniferyl alcohol lignin model compound (dehydrogenate polymer a.k.a. ZL-DHP) at the air-water interface. Using the CHCl(3)/MeOH (5:1 v/v) spreading solvent, we found an average molecular area of ZL-DHP of approximately 1200 A(2). The monolayer expresses a high compressibility with a collapsed area of 500 A(2) and collapsed surface pressure of 28 mN m(-1). In the range of applied surface pressures, ZL-DHP polymer have no phase changes, as shown by the very high linearity (R=0.994) of absorbance vs. surface pressure cure. There was no symmetry transitions observed as shown by absence of shifts of absorption peak maximums.

Two amphiphilic PAMAM dendrimers are synthesized by attaching 12-hydroxydodecanoic acid (HA) chains to a poly(amido amine) (PAMAM) dendrimer core (including generation I and generation II). The limiting molecular area obtained from the surface pressure-area isotherm at the air/water interface suggests the edge-on configuration for both dendrimers in Langmuir films. The edge-on arrangement is also supported by the atomic force microscopic (AFM) studies of the Langmuir-Blodgett films.

By combining the results from atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM), herein we investigate properties of photochemical lignin model compounds. We provide evidence that photochemical lignin forms random, probably non-functional structures. The topography of such structures is explored using ESEM. Non-functionality of such structures is proved by AFM and atomic force spectroscopy experiments wherein the photochemical lignin functionalized tip is approached to the substrate covered with photochemical lignin. There was no evidence of existence of any kind of host-guest interaction during the approach/retraction experiments. These results provide evidence for our previously stated hypothesis that photochemical lignin polymerization may be one of the degrading effects of UV radiation to the plant cell.

Environmental scanning electron microscopy images of the self-assembled structures of enzymatically (DHP) and photochemically polymerized (PCP) artificial lignin are herein presented. Differences in the structural organization between DHP and PCP polymer at the supramolecular level were reported. Based on topological information, we proposed a hypothesis about possible new physiological roles of lignin in live plant cells and the ecological significance of possible in-vivo photochemical lignin polymerization. Copyright 2000 Academic Press.

In this paper we are presenting the results of our environmental scanning electron microscopy (ESEM) investigation of the lignin model compound--enzymatically polymerized coniferyl alcohol, also known as dehydrogenate polymer (DHP). The goals of this study were to visualize the supramolecular organization of DHP polymer on various substrates, namely graphite, mica, and glass, and to explore the influence of substrate surface properties and associated collective phenomena on the lignin self-assembled supramolecular structure. Based on results obtained with ESEM, combined with previously published results based on scanning tunneling microscopy (STM) and electron spin resonance (ESR) technique, we looked at lignin structure ranging from a monomer on a fraction of nanometer scale to a large aggregate on a fraction of millimeter scale, therefore using six orders of magnitude range of size. Herein, we are presenting evidence that there are at least four different levels of the supramolecular structure of lignin, and that its supramolecular organization is well dependent on the substrate surface characteristics, such as hydrophobicity, delocalized orbitals, and surface-free energy.

Protein-functionalized atomic force microscopy (AFM) tips have been used to investigate the interaction of individual ligand-receptor complexes. Herein we present results from scanning electron microscopy (SEM) studies of protein-functionalized AFM cantilever tips. The goals of this study were (1) to examine the surface morphology of protein-coated AFM tips and (2) to determine the stability of the coated tips. Based on SEM images, we found that bovine serum albumin (BSA) in solution spontaneously adsorbed onto the surface of silicon nitride cantilevers, forming a uniform protein layer over the surface. Additional protein layers deposited over the initial BSA-coated surface did not significantly alter the surface morphology. However, we found that avidin-functionalized tips were contaminated with debris after a series of force measurements with biotinylated agarose beads. The bound debris presumably originated from the transfer of material from the agarose bead. This observation is consistent with the observed deterioration of functional activity as measured in ligand-receptor binding force experiments.

To assess the significance of gonadal function in the maintenance of thymic structure, male, sexually mature, 60 days old rats were bilaterally orchidectomized and the volumes of both the thymic compartments, total cellularity of each of them, as well as density (Nv) and mean diameter (D) of thymocytes within the specific thymic regions were stereologically analyzed 3, 7 and 15 days later. Three days after the orchidectomy a decrease in the thymic weight, and a reduction of both the cortical and medullary volumes, were observed. The reduction in cortical volume was followed by a decrease in overall number of the cortical thymocytes, suggesting an enhanced process of thymocyte death. In the same animals, the total number of the medullary thymocytes as well as D remained unchanged. Seven days after orchidectomy both volume of the cortex and total number of the cortical thymocytes reached the control values, while all the changes observed in the medulla of rats 3 days post castration, persisted. In addition, 7 days post castration, the volume of the interlobular connective tissue was increased and this persisted at day 15 post castration. The results indicate that orchidectomy can affect the thymic structure, and possibly the process of intrathymic T cell maturation; the extent of changes depending on the duration of the gonadal deprivation.

To assess the gonadal influence on the maturation of the thymus, rats were orchidectomized at different periods critical for programming of both gonadal and immune functions, and the composition of the intrathymic thymocyte population was determined in adults by flow cytometric analysis of the surface phenotype. The relative proportion of thymocytes expressing CD4, CD8 and T cell receptor (TCR)alpha beta was measured in the adult rats castrated at 1, 7 and 30 days. Castration performed at postnatal day 1 did not significantly affect expression of these molecules. However, in rats subjected to the surgery at day 7, a significant decrease in the proportion of CD4+CD8+ double-positive cells was found, in parallel with a proportional increase in the percentage of CD4+CD8- single-positive (SP) cells. In rats castrated at age 30 days, in addition to these changes, a small but significant increase in the percentage of CD4-CD8+ SP thymocytes was measured. Castration performed at age 7 and 30 days also caused an enrichment in the thymocyte population expressing TCR alpha beta, probably related to the increase of CD4+CD8- SP cells (7 days) and both CD4+CD8- and CD4-CD8+ SP cells (30 days). The total yield of thymocytes was increased in all experimental groups. We conclude that T cell maturation sequences in the adult thymus are altered if gonadal influence is removed during certain stages of thymus development.

Clinical and meiotic studies were done on an infertile man with a translocation between Yq and 13q, who was identified through the birth of his son with partial trisomy 13q. Seminal plasma transferrin showed preserved Sertoli cell function while lactate dehydrogenase C4 indicated hypospermatogenesis. A quadrivalent in diakinesis and spermatogenic arrest in the second meiotic division was detected.

A meiotic analysis in a group of 90 infertile men was carried out and also histological examination. In one third of patients there were no cells in divisions and Sertoli cells were only seen in most of them. In 30% of patients the process of spermatogenesis stopped at pachytene or between the first and second meiotic division. In 38% of patients all stages of spermatogenesis and of meiosis were present. Chromosomal translocations were detected in three patients in diakinesis. The testicular biopsy is an important tool in diagnosis and management of male infertility. The histological and meiotic investigations of the testicular tissue specimen give us the information obtainable in no other way. These both investigations are essential for prognosis and treatment of infertile men.