An unusual presentation of primary cutaneous follicle center lymphoma.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare low-grade cutaneous B-cell lymphoma. Clinically, PCFCL is usually an erythematous subcutaneous nodule or an infiltrated plaque. The dermoscopy is non-specific and it is characterized by polymorphous vascular pattern, arborizing vessels over a salmon-colored background and white areas. We reported a case of a 36-year-old woman presented with a rapidly growing, flashed-color, exophytic, soft consistency nodule on her scalp. Dermoscopy showed a diffuse structureless, skin-color area associated with a rare arborizing vascular pattern and brown circles. We reported a peculiar clinical and dermoscopic variant. This clinical presentation of PCFCL is unusual and represents a pitfall in the early clinical diagnosis. Histopathology is mandatory for a correct diagnosis.

Dupilumab therapy of atopic dermatitis of the elderly: a multicentre, real-life study.

BACKGROUND: Treatment of moderate-to-severe atopic dermatitis (AD) in the elderly may be challenging, due to side-effects of traditional anti-inflammatory drugs and to comorbidities often found in this age group. Furthermore, efficacy and safety of innovative drugs such as dupilumab are not yet well known. OBJECTIVES: A multicentre retrospective, observational, real-life study on the efficacy and safety of dupilumab was conducted in a group of patients aged ≥65 years and affected by severe AD. Their main clinical features were also examined. METHODS: Data of elderly patients with severe (EASI ≥24) AD treated with dupilumab at label dosage for 16 weeks were retrospectively collected. Treatment outcome was assessed by comparing objective (EASI) and subjective (P-NRS, S-NRS and DLQI) scores at baseline and after 16 weeks of treatment. RESULTS: Two hundred and seventy-six patients were enrolled in the study. They represented 11.37% of all patients with severe AD. Flexural eczema was the most frequent clinical phenotype, followed by prurigo nodularis. The coexistence of more than one phenotype was found in 63/276 (22.82%) subjects. Data on the 16-week treatment with dupilumab were available for 253 (91.67%) patients. Efficacy of dupilumab was demonstrated by a significant reduction of all the scores. No statistically significant difference regarding efficacy was found in elderly patients when compared to the group of our AD patients aged 18-64 years, treated with dupilumab over the same period. Furthermore, only 18 (6.52%) patients discontinued the drug due to inefficacy. Sixty-one (22.51%) patients reported adverse events, conjunctivitis and flushing being the most frequent. One (0.36%) patient only discontinued dupilumab due to an adverse event. CONCLUSIONS: Therapy with dupilumab led to a significant improvement of AD over a 16-week treatment period, with a good safety profile. Therefore, dupilumab could be considered as an efficacious and safe treatment for AD also in the elderly.

Engineering approaches for drug delivery systems production and characterization.

To find and to test the therapeutic effectiveness (and the limited adverse effects) of a new drug is a long and expensive process. It has been estimated a period of ten years and an expense of the order of one billion USD are required. Meanwhile, even if a promising molecule has been identified, there is the need for operative methods for its delivery. The extreme case is given by gene therapy, in which molecules with tremendous in-vitro efficacy cannot be used in practice because of the lack in useful vector systems to deliver them. Most of the recent efforts in pharmaceutical sciences are focused on the development of novel drug delivery systems (DDSs). In this review, the work done recently on the development and testing of novel DDSs, with particular emphasis on the results obtained by European research, is summarized. In the first section of the review the DDSs are analyzed accordingly with their scale-size: starting from nano-scale (liposomes, nanoparticles), up to the micro-scale (microparticles), until the macroscopic world is reached (granules, matrix systems). In the following two sections, non-conventional testing methods (mechanical methods and bio-relevant dissolution methods) are presented; at last, the importance of mathematical modeling to describe drug release and related phenomena is reported.

Pilonidal sinus disease: an intergluteal localization of hidradenitis suppurativa/acne inversa: a cross-sectional study among 2465 patients.

BACKGROUND: Hidradenitis suppurativa (HS), also referred to as acne inversa, is a debilitating skin disease characterized by inflammatory nodules, chronic abscesses and tunnels (fistulae and sinuses). The association with pilonidal sinus disease (PSD) is frequently reported but not well documented. OBJECTIVES: To determine the prevalence and characteristics of inflammatory skin lesions located in the intergluteal fold (IGF) of patients with HS. METHODS: This was an international multicentre retrospective cross-sectional study based on data collection from a large cohort of patients with HS with and without histopathology. Results From a total of 2465 patients with HS included in the study, 661 (27%) reported lesions in the IGF. These patients were significantly more often smokers and had more severe HS. Of the 238 patients with an available clinical diagnosis, intergluteal-HS (IG-HS) was diagnosed in 52 patients (22%) and PSD was diagnosed in 186 patients (78%). IG-HS was associated with the localization of HS in the proximity of the IGF, including the buttocks, genitals and the anus. There was a possibility of misclassification bias in this study as a clinical/image-based diagnosis or histopathology of the IGF lesions was not always available. CONCLUSIONS: The high prevalence of PSD suggests a strong link between both entities. Therefore, it may be useful to identify common pathophysiological mechanisms and develop common therapeutic strategies. What's already known about this topic? The occurrence of pilonidal sinus disease has not been clearly reported among patients with hidradenitis suppurativa/acne inversa. What does this study add? This is the first study that investigated the prevalence of pilonidal sinus disease among a large cohort of patients and identified the patient characteristics. Risk factors that might help to improve the management of patients were identified.

Human cardiac multipotent adult stem cells in 3D matrix: new approach of tissue engineering in cardiac regeneration post-infarction.

Myocardial infarction is the leading cause of morbidity and mortality in developed countries. It causes a left ventricular dysfunction, mainly due to the loss of functional tissue, resulting in heart failure. New therapies are being developed, using a tissue engineering approach, with the ultimate goal of restoring cardiac function by regenerating and repairing the damaged myocardium. In the present study we investigated the behaviour of a specific population of c-kit positive human cardiac stem cells, called Multipotent Adult Stem Cells (MASCs), grown within three-dimensional collagen scaffolds (3D), to establish whether they could be used in post-infarction cardiac regeneration. We also evaluated the expression levels of the Granulocyte Macrophage-Colony Stimulating Factor Receptor (GM-CSFR) and endoglin, a component of the Transforming Growth Factor beta (TGF-ß) receptor complex. Finally, we also evaluated the expression of the α2ß1integrin. MASCs cultured within 3D collagen matrices are able to proliferate and migrate even in the absence of chemotactic agents and express high levels of factors involved in cell proliferation and migration, such as GM-CSFRα chain and integrins. They therefore represent a promising approach to tissue engineering aimed to restore cardiac function. Our results also suggest a role of GM-CSF in cell proliferation, while TGF-ß does not seem to be relevant.

Lipid-lowering effect of bergamot polyphenolic fraction: role of pancreatic cholesterol ester hydrolase.

Bergamot polyphenolic fraction (BPF) has been shown to positively modulate several mechanisms involved in metabolic syndrome, suggesting its use in therapy. In particular, it is able to induce a significant amelioration of serum lipid profile in hyperlipemic patients at different levels. The purpose of our study was to investigate the effect of BPF on cholesterol absorption physiologically mediated by pancreatic cholesterol ester hydrolase (pCEH). An in vitro activity assay was performed to study the effect of BPF on pCEH, whereas the rate of cholesterol absorption was evaluated through in vivo studies. In particular, male, Sprague-Dawley rats (200–225 g) were fed either normal chow or chow supplemented with 0.5% cholic acid, 5.5% peanut oil, and varying amounts of cholesterol (0 to 1.5%). BPF (10 mg/Kg) was daily administrated by means of a gastric gavage to animals fed with lipid supplemented diet for 4 weeks and, at the end of the study, plasma lipids and liver cholesteryl esters were measured in all experimental groups. Our results show that BPF was able to inhibit pCEH activity and this effect was confirmed, in vivo, via detection of lymphatic cholesteryl ester in rats fed with a cholesterol-rich diet. This evidence clarifies a further mechanism responsible for the hypolipemic properties of BPF previously observed in humans, confirming its beneficial effect in the therapy of hypercholesterolemia and in the treatment of metabolic syndrome.

A novel versatile precursor suitable for 18 F-radiolabeling via "click chemistry".

As an effort to improve 18 F-radiolabeling of biomolecules in method robustness and versatility, we report the synthesis and radiolabeling of a new azido precursor potentially useful for the so-called "click reaction," in particular the ligand-free version of the copper(I)-catalyzed alkyne-azide cycloaddition. The new azido precursor may help to overcome problems sometimes exhibited by most of the currently used analogues, as it is safe to handle and it displays long-term chemical stability, thus facilitating the development of new radiolabeling procedures. Moreover, the formed 18 F-labeled 1,2,3-triazole is potentially metabolically stable and could enhance the in vivo circulation time. The above azido precursor was successfully radiolabeled with 18 F, with 51% radiochemical yield (nondecay-corrected). As a proof of concept, the 18 F-labeled azide was then tested with a suitable alkyne functionalized aminoacid (l-propargylglycine), showing 94% of conversion, and a final radiochemical yield of 27% (>99% radiochemical purity), nondecay-corrected, with a total preparation time of 104 minutes.

Contact dermatitis in patients with chronic leg ulcers: a common and neglected problem: a review 2000-2015.

OBJECTIVE: Contact allergies can occur frequently in patients with chronic leg ulcers (CLUs), even in those with a short duration of ulcerative disease. The wide spectrum of therapeutic products promotes development of the delayed type of hypersensitivity and continuous changes in the allergens pattern, which make the diagnosis and treatment extremely difficult in many cases. A prompt diagnosis and treatment of allergic contact dermatitis (ACD) in patients suffering from CLUs is very important for a best clinical outcome of these two common diseases. Thus, this review aims to highlight a common, challenging and often neglected problem. METHOD: The search included all studies published between 2000 and September 2015. Keyword used were: 'allergic contact dermatitis leg ulcer', 'contact dermatitis leg ulcers' 'contact dermatitis wound care' 'contact dermatitis non-healing wounds' 'contact sensitisation non-healing wounds'. RESULTS: Contact allergy and polysensitisation are very frequent in patients suffering from CLUs. Although it is believed modern dressings have a lower potential for inducing cutaneous sensitisation, positive patch test reactions to modern dressing are becoming common: hydrogels, followed by hydrocolloid and the ionic silver-containing wound dressing seem to be the principal causes of ACD. CONCLUSION: This review wanted to highlighted ACD in CLUs as a common and neglected disease whose economic and social burden has not previously been estimated, giving new insights for clinical and therapeutic management.

Optimizing design and fabrication of microfluidic devices for cell cultures: An effective approach to control cell microenvironment in three dimensions.

The effects of gradients of bioactive molecules on the cell microenvironment are crucial in several biological processes, such as chemotaxis, angiogenesis, and tumor progression. The elucidation of the basic mechanisms regulating cell responses to gradients requires a tight control of the spatio-temporal features of such gradients. Microfluidics integrating 3D gels are useful tools to fulfill this requirement. However, even tiny flaws in the design or in the fabrication process may severely impair microenvironmental control, thus leading to inconsistent results. Here, we report a sequence of actions aimed at the design and fabrication of a reliable and robust microfluidic device integrated with collagen gel for cell culturing in 3D, subjected to a predetermined gradient of biomolecular signals. In particular, we developed a simple and effective solution to the frequently occurring technical problems of gas bubble formation and 3D matrix collapsing or detaching from the walls. The device here proposed, in Polydimethylsiloxane, was designed to improve the stability of the cell-laden hydrogel, where bubble deprived conditioning media flow laterally to the gel. We report the correct procedure to fill the device with the cell populated gel avoiding the entrapment of gas bubbles, yet maintaining cell viability. Numerical simulations and experiments with fluorescent probes demonstrated the establishment and stability of a concentration gradient across the gel. Finally, chemotaxis experiments of human Mesenchymal Stem Cells under the effects of Bone Morphogenetic Protein-2 gradients were performed in order to demonstrate the efficacy of the system in controlling cell microenvironment. The proposed procedure is sufficiently versatile and simple to be used also for different device geometries or experimental setups.

Prevalence of depression in adults with haemophilia.

Multiple factors place adults with haemophilia at risk for depression. Health outcomes can be compromised in depressed patients secondary to increased risk taking behaviour and poor compliance with treatment recommendations. To assess the prevalence and risk factors associated with depression in adult patients with haemophilia treated at a haemophilia treatment centre. Adults with haemophilia were screened for depression during their annual clinic visit using the Patient Health Questionnaire 9 (PHQ-9), a validated tool for depression screening in adults. Depression was defined as a PHQ-9 score ≥ 5. Risk factors associated with depression were collected by chart review and correlated with depression scores. A total of 41 adult patients consented to the study and 37% met criteria for depression. Fifty-three per cent of patients with depression reported moderate to severe symptoms of depression (PHQ-9 score >10). Seventy-six per cent of patients with depression reported suffering functional impairment due to their depressive symptoms. Lack of social support and unemployment were significantly associated with higher PHQ-9 scores (P = 0.04 and P = 0.01 respectively). Adult patients with haemophilia have a high prevalence of depression. The addition of depression screening to the comprehensive care of adults with haemophilia may result in improved overall health outcomes and treatment adherence.

Engineered mu-bimodal poly(epsilon-caprolactone) porous scaffold for enhanced hMSC colonization and proliferation.

The use of scaffold-based strategies in the regeneration of biological tissues requires that the design of the microarchitecture of the scaffold satisfy key microstructural and biological requirements. Here, we examined the ability of a porous poly(epsilon-caprolactone) (PCL) scaffold with novel bimodal-micron scale (mu-bimodal) porous architecture to promote and guide the in vitro adhesion, proliferation and three-dimensional (3-D) colonization of human mesenchymal stem cells (hMSCs). The mu-bimodal PCL scaffold was prepared by a combination of gas foaming (GF) and selective polymer extraction (PE) from co-continuous blends. The microarchitectural properties of the scaffold, in particular its morphology, porosity distribution and mechanical compression properties, were analyzed and correlated with the results of the in vitro cell-scaffold interaction study, carried out for 21days under static conditions. Alamar Blue assay, scanning electron microscopy, confocal laser scanning microscopy and histological analyses were performed to assess hMSC adhesion, proliferation and 3-D colonization. The results showed that the combined GF-PE technique allowed the preparation of PCL scaffold with a unique multiscaled and highly interconnected microarchitecture that was characterized by mechanical properties suitable for load-bearing applications. Study of the cell-scaffold interaction also demonstrated the ability of the scaffold to support hMSC adhesion and proliferation, as well as the possibility to promote and guide 3-D cell colonization by appropriately designing the microarchitectural features of the scaffold.

Nitric oxide, polyamines and Cd-induced phytotoxicity in wheat roots.

To further explore the biochemical basis of Cd toxicity in developing wheat seedlings, we studied the possible role of nitric oxide (NO) and polyamines as signaling molecules involved in metal-induced root growth inhibition. When used at 0.1 mM, sodium nitroprusside, a NO-releasing compound, inhibited root growth to a similar extent as Cd and enhanced the polyamine contents as Cd also did. Putrescine and spermidine treatments caused significant decreases in root growth with spermine giving the greatest level of inhibition (77% reduction). The simultaneous addition of Cd and inhibitors of putrescine biosynthesis (DFMA and DFMO) prevented increases in putrescine levels but did not restore normal root growth. NO content, as evidenced by the fluorescent probe DAF-FM diacetate, was found to be significantly increased in the roots of both Cd and polyamine treated plants, especially in those exposed to spermine. The effect was specific for NO since the NO scavenger cPTIO almost suppressed the fluorescent signal. Concerning the oxidative status of the root system, only Cd and spermine enhanced lipid peroxidation in roots. At the same time, all treatments led to a significant increase in levels of the non-enzymatic antioxidant defense glutathione. Our results strongly suggest that Cd and spermine treatments induce NO formation in wheat roots which, in turn, is involved in root growth inhibition.

No evidence of involvement of Chlamydia pneumoniae in lung cancer by means of quantitative real-time polymerase chain reaction.

Chlamydia pneumoniae, an obligate intracellular pathogen, is well-known as etiological agent of acute respiratory infections; the repeated or prolonged exposure to chlamydial antigens may promote the persistence of C. pneumoniae in the respiratory tract leading to chronic diseases, such as chronic obstructive pulmonary disease and asthma. The predilection of C. pneumoniae to cause respiratory tract infections combined with its persistent nature suggest that it might play a role in lung cancer. The aim of our study is to evaluate the involvement of C. pneumoniae in pathogenesis of lung cancer. We therefore investigated the presence of C. pneumoniae DNA in tumor lung tissues by using real-time PCR assay. Simultaneously, tumor and healthy tissues from the same patient with primary carcinoma lung were analyzed. C. pneumoniae DNA was not detected in a single lung tumor tissue by means of an highly sensitive, and specific real-time PCR assay based on FRET hybridization probes. In conclusion, this study does not support the involvement of C. pneumoniae in the pathogenesis of lung cancer, suggesting that further investigations are needed to clarify other potential causative factors for the development of this malignancy.

Molecular modelling and enzymatic studies of acetylcholinesterase and butyrylcholinesterase recognition with paraquat and related compounds.

The potent herbicide paraquat and three other analogues MPP+, MPDP+ and MPTP have a known toxicological profile linked to the ability to damage dopaminergic neurons. Other biological effects were recently addressed to this class of compounds, including the ability to interact with enzymatic targets involved in the Central Nervous System, such as the acetylcholinesterase (AChE) and the butyrylcholinesterase (BuChE). A combined molecular modelling and enzymatic study focusing onto their interaction against the AChE and BuChE is reported. The former study was performed by docking techniques using target known co-crystallographic models. The latter study was carried out by the widely adopted Ellman's method. In both studies the anti-Alzheimer FDA approved drug tacrine was used as reference inhibitor. Our results indicate that paraquat, MPTP, MPDP+ and MPP+ recognize both enzymatic cleft in a similar fashion compared to the reference inhibitor. A structure-activity correlation was found with the net charge of the ligands, indicating a major role of the electrostatic term in the recognition and inhibition of these compounds. Our data completed their enzymatic profile, added new information on the molecular mechanisms underlying their neurotoxicity useful for the rational design of new cholinesterase inhibitors.

Central cardiovascular responses induced by interleukin 1 beta and tumor necrosis factor alpha infused into nucleus tractus solitarii, nucleus parabrachialis medialis and third cerebral ventricle of normotensive rats.

The effect of IL-1 beta and TNF alpha infused into nucleus tractus solitari (NTS), nucleus parabrachialis medialis (NPBmed) and third cerebral ventricle of normotensive rats on blood pressure (BP) and heart rate (HR) was investigated. Microinfusion of IL-1 beta and TNF alpha into the third cerebral ventricle and NPBmed of normotensive rats produced a dose-dependent hypotensive and bradycardic response. A similar cardiovascular response was produced by infusion of IL1 beta into NTS but not by TNF alpha. When rats were pre-treated with Escherichia coli lipopolisaccharide (LPS), an enhancement of cardiovascular response elicited by IL-1 beta and TNF alpha was found. Thus, IL-1 beta and TNF alpha produce cardiovascular responses when infused into specific areas of the CNS. This effect is potentiated by LPS and this may explain the alteration in cardiovascular regulation which can be observed in diseases in which an excess of circulating endotoxins and cytokines may occur.

Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres.

BACKGROUND: We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins. METHODS: In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence. RESULTS: We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen). CONCLUSIONS: This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding.

Disruption of the EF-2 kinase/Hsp90 protein complex: a possible mechanism to inhibit glioblastoma by geldanamycin.

Glioblastoma multiforme is the most treatment-resistant brain tumor. Elongation factor-2 (EF-2) kinase (calmodulin kinase III) is a unique protein kinase that is overexpressed in glioma cell lines and in human surgical specimens. Several mitogens activate this kinase and inhibitors block mitogen activation and produce cell death. Geldanamycin (GA) is a benzoquinone ansamycin antibiotic that disrupts Hsp90-protein interactions. Because EF-2 kinase is chaperoned by Hsp90, we investigated the effects of GA on the viability of glioma cells, the expression of EF-2 kinase protein, and the interaction between Hsp90 and EF-2 kinase. GA was a potent inhibitor of the clonogenicity of four glioma cells lines with IC(50)s ranging from 1 to 3 nM. 17-allylamino-17-demethoxygeldanamycin (17-AAG), a less toxic and less potent derivative of GA, inhibited the clonogenicity of glioma cells with IC(50) values of 13 nM in C6 cells and 35 nM in T98G cells. Treatment of cell lines for 24-48 h of GA or 17-AAG disrupted EF-2-kinase/Hsp90 interactions as measured by coimmunoprecipitation, resulting in a decreased amount of recoverable kinase in cell lysates. The ability of GA to inhibit the growth of glioma cells was abrogated by overexpressing EF-2 kinase. In addition, 17-AAG significantly inhibited the growth of a glioma xenograft in nude mice. These studies demonstrate for the first time the activity of GAs against human gliomas in vitro and in vivo and suggest that destruction of EF-2 kinase may be an important cytotoxic mechanism of this unique class of drug.

Flow cytometric platform for high-throughput single nucleotide polymorphism analysis.

We have developed a rapid, cost-effective, high-throughput readout for single nucleotide polymorphism (SNP) genotyping using flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs a PCR-derived target DNA containing the SNP, a synthetic SNP-complementary ZipCode-bearing capture probe, a fluorescent reporter molecule, and a thermophilic DNA polymerase. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to the reaction products and sequestered them for flow cytometric analysis. The single base chain extension (SBCE) reaction was used to assay 20 multiplexed SNPs for 633 patients in 96-well format. Comparison of the microsphere-based SBCE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.3% agreement in genotype assignments. Substitution of direct-labeled R6G dideoxynucleotide with indirect-labeled phycoerythrin dideoxynucleotide enhanced signal five- to tenfold while maintaining low noise levels. A new assay based on allele-specific primer extension (ASPE) was validated on a set of 15 multiplexed SNPs for 96 patients. ASPE offers both the advantage of streamlining the SNP analysis protocol and the ability to perform multiplex SNP analysis on any mixture of allelic variants.

Microsphere-based molecular cytometry.

Flow cytometry has recently gained attention as a powerful high-throughput technology for the analysis of molecular interactions. This recognition is mostly because of the development and use of fluorescently distinct microsphere populations. Recent applications of this technology have included detecting analytes, monitoring enzymatic activity, and genotyping SNP. This article expands the list of applications by highlighting nuclear receptor-coactivator peptide binding studies. Nuclear receptors initiate gene transcription by binding to a wide variety of accessory factors. Small molecule ligand binding by nuclear receptors modulates the interaction with some of these cofactors. Because a typical cell contains numerous cofactors, multiplexed analysis of nuclear receptor binding to coactivator peptide-coupled microspheres is a valuable approach to rapidly assess this network of complex interactions. Understanding how ligand binding regulates these interactions should help in the design of improved small molecule therapeutics. The latter part of this article has focused on the recent application of multiplexing protein:protein interactions. Developing protein interaction networks will be paramount as a greater understanding of the human genome is developed. In the future, flow cytometric analysis of fluorescent microspheres may be applied to any array of interacting molecules and may have broader applications in the area of proteomics.

Expression, purification, and characterization of the human receptor activator of NF-kappaB ligand (RANKL) extracellular domain.

Receptor activator of NF-kappaB ligand (RANKL) is a type II transmembrane protein found on osteoblasts which functions as a major determinant of osteoclast differentiation and activation. RANKL mediates bone homeostasis through binding to the cognate ligand on osteoclasts, RANK, and a soluble decoy receptor, osteoprotegerin (OPG). We designed a construct encoding the extracellular domain of human RANKL that conformed to reports of native processing. To encourage folding and posttranslational modification of a normally membrane-inserted moiety, we expressed the RANKL truncate as a secreted protein using the signal sequence from OPG in a Trichoplusia ni cell line using a baculovirus expression vector. RANKL was purified by a three-step process including an OPG-Fc affinity column. SDS-PAGE and mass spectral analysis indicated that the protein was >99% pure and glycosylated. Circular dichroism spectra revealed that the protein exhibited structural elements similar to tumor necrosis factor-alpha. By BIAcore analysis, RANKL bound to OPG with an affinity of 6.7 nM. Sedimentation equilibrium analytical ultracentrifugation analyses established that our protein existed as a trimer. We conclude that our expressed human RANKL truncate is folded, is functional, and exhibits self-association consistent with other family members.