Isolation of biofluids from tissues using a vacuum-assisted filtration biomedical device.

A biopsy is usually used to remove a piece of tissue from a patient for laboratory testing. The interstitial fluid is taken out at the same time as the tissue sample. Since interstitial fluid flows between cells and capillaries in tissues, similar to blood plasma, it is necessary to separate interstitial fluid from tissues in order to study them separately. Vacuum blood sampling has been used to draw blood into vacuum-sealed tubes, while interstitial fluid can be removed directly from the skin using microneedles with standard pumps. However, no methods are available to separate blood or interstitial fluid from the tissue itself for molecular characterization. In this study, we designed a biomedical device that can separate interstitial fluid from tissue using a vacuum-assisted filtration method. The device has a chamber that collects fluid extracted from the tissue that remains on top of the filter. We characterized the weight change and glycan profiles of tissues before and after vacuum-assisted filtration. The results demonstrate that the biomedical device can remove interstitial fluid and facilitate the analysis of tissue-specific molecules while minimizing information from the interstitial fluid.

Protein glycosylation in urine as a biomarker of diseases.

Human body fluids have become an indispensable resource for clinical research, diagnosis and prognosis. Urine is widely used to discover disease-specific glycoprotein biomarkers because of its recurrently non-invasive collection and disease-indicating properties. While urine is an unstable fluid in that its composition changes with ingested nutrients and further as it is excreted through micturition, urinary proteins are more stable and their abnormal glycosylation is associated with diseases. It is known that aberrant glycosylation can define tumor malignancy and indicate disease initiation and progression. However, a thorough and translational survey of urinary glycosylation in diseases has not been performed. In this article, we evaluate the clinical applications of urine, introduce methods for urine glycosylation analysis, and discuss urine glycoprotein biomarkers. We emphasize the importance of mining urinary glycoproteins and searching for disease-specific glycosylation in various diseases (including cancer, neurodegenerative diseases, diabetes, and viral infections). With advances in mass spectrometry-based glycomics/glycoproteomics/glycopeptidomics, characterization of disease-specific glycosylation will optimistically lead to the discovery of disease-related urinary biomarkers with better sensitivity and specificity in the near future.

Effect of Carbon Chain Length, Ionic Strength, and pH on the In Vitro Release Kinetics of Cationic Drugs from Fatty-Acid-Loaded Contact Lenses.

This paper explores the use of fatty acids in silicone hydrogel contact lenses for extending the release duration of cationic drugs. Drug release kinetics was dependent on the carbon chain length of the fatty acid loaded in the lens, with 12-, 14- and 18-carbon chain length fatty acids increasing the uptake and the release duration of ketotifen fumarate (KTF) and tetracaine hydrochloride (THCL). Drug release kinetics from oleic acid-loaded lenses was evaluated in phosphate buffer saline (PBS) at different ionic strengths (I = 167, 500, 1665 mM); the release duration of KTF and THCL was decreased with increasing ionic strength of the release medium. Furthermore, the release of KTF and THCL in deionized water did not show a burst and was significantly slower compared to that in PBS. The release kinetics of KTF and THCL was significantly faster when the pH of the release medium was decreased from 7.4 towards 5.5 because of the decrease in the relative amounts of oleate anions in the lens mostly populated at the polymer-pore interfaces. The use of boundary charges at the polymer-pore interfaces of a contact lens to enhance drug partition and extend its release is further confirmed by loading cationic phytosphingosine in contact lenses to attract an anionic drug.

Evaluation of commercial soft contact lenses for ocular drug delivery: A review.

Soft contact lenses have generated growing interest in ocular drug delivery due to their potential to enhance drug bioavailability in ocular tissues. Commercially available soft contact lenses offer several advantages for ocular drug delivery as they are manufactured on a large scale, which guarantees the availability of a consistent and reproducible product, and their favorable safety profile is well-established through broad clinical use. Here we review the rationale for using commercially available soft contact lenses for ocular drug delivery; summarize the evolution of the materials used in contact lens fabrication; and explore various methods used to improve the drug release characteristics and its tissue penetration. While significant progress has been made, several issues still require further attention for the commercial launch of a viable drug-eluting contact lens product, including control of initial burst release, shelf-life stability, and drug loss during processing or storage.

Extended delivery of cationic drugs from contact lenses loaded with unsaturated fatty acids.

This paper describes the use of surface-active anionic unsaturated fatty acids in commercial contact lenses to extend drug release duration and regulate delivery dosage. We studied the effect of oleic acid on the in vitro release kinetics of three cationic drugs, and two anionic drugs from silicone hydrogel contact lenses. The release duration of the cationic drugs: tetracaine hydrochloride, bupivacaine hydrochloride, and ketotifen fumarate was significantly extended from less than a day to more than a month because of the presence of oleic acid in the contact lenses. With a simple change in the fatty acid loading media, we could duplicate a similar efficacy by loading oleic acid in conventional non-silicone hydrogel contact lenses. The fitted effective diffusivity values of the three cationic drugs significantly decrease when the oleic acid weight % in the lenses is increased. By using two other unsaturated fatty acids, linoleic and α-linolenic acid, the release duration of ketotifen fumarate was also significantly extended in silicone hydrogel contact lenses. In contrast, the release of two anionic drugs, diclofenac sodium and flurbiprofen sodium, was accelerated for oleic acid modified lenses. These results show the dominating impact of coupling charge interactions between the drug and the fatty acid carrier molecules to precisely adjust delivery rate and dosage from a contact lens.

Hydrogel-based ocular drug delivery systems for hydrophobic drugs.

Conventional ophthalmic dosage forms such as eye drops pose a significant challenge because physiological barriers and clearance mechanisms limit ocular bioavailability. Hydrogels are promising therapeutic materials for ocular drug delivery because of their high biocompatibility and their ability to hold and release therapeutic agents. Even though they are generally associated with the delivery of hydrophilic drugs, several approaches have been developed to integrate hydrophobic ophthalmic drugs into hydrogels. Because of the limitations associated with the traditional topical eye drop delivery of hydrophobic drugs, hydrogel-based systems represent a viable alternative for controlled ocular drug delivery. This review presents an overview on the ophthalmic applications of hydrogels for the delivery of hydrophobic drugs, with special focus on diseases occurring in the anterior segment of the eye. We summarize the key hydrogels for incorporation and delivery of hydrophobic drugs, including soft contact lenses (SCLs), stimuli-responsive hydrogels, cyclodextrin-based polymeric hydrogels, and nanoparticle-loaded hydrogels. The strategies of integrating hydrophobic drugs into hydrogels as discussed in this review provide significant potential in ocular therapeutics.

Closer to the Truth on National Fistula Prevalence After Unilateral Complete Cleft Lip and Palate Repair? The Cleft Care UK Study.

OBJECTIVES: To (1) determine the prevalence of nonperialveolar palatal fistula up to age 5 following repair of unilateral cleft lip and palate (UCLP) in the United Kingdom, (2) examine the association of palatoplasty techniques with fistula occurrence, and (3) describe the frequency of fistula repairs and their success. DESIGN: Cross-sectional study. SETTING: All 11 centralized regional cleft centers in the United Kingdom. PARTICIPANTS: Two hundred sixty-eight children born between 2005 and 2007 recruited by Cleft Care UK, a nationwide cross-sectional study of all 5-year-old children born with nonsyndromic UCLP. MAIN OUTCOME MEASURE: Nonperialveolar palatal fistula prevalence up to age 5. RESULTS: Fistulas were found in 72 children (31.3%, 95% confidence interval: 25.4%-37.7%) and had no significant association with palate repair sequences. Twenty-four fistulas were repaired by age 5, 12 of which had data showing 10 (83.3%) successful repairs. CONCLUSION: The prevalence of nonperialveolar fistulas following primary palatoplasty of UCLP in the United Kingdom was higher than previously reported. This information should be part of the preoperative discussion with families. Prospective collection of the presence of fistulas will be necessary before we can associate the occurrence of fistulas with a surgeon, institution, surgical technique, or protocol of care.

Formation of Drug-Participating Catanionic Aggregates for Extended Delivery of Non-Steroidal Anti-Inflammatory Drugs from Contact Lenses.

This paper focuses on extending drug release duration from contact lenses by incorporating catanionic aggregates. The aggregates consist of a long-chain cationic surfactant, i.e., cetalkonium chloride (CKC), and an oppositely charged anti-inflammatory amphiphilic drug. We studied three non-steroidal anti-inflammatory (NSAID) drugs with different octanol-water partition coefficients; diclofenac sodium (DFNa), flurbiprofen sodium (FBNa), and naproxen sodium (NPNa). Confirmation of catanionic aggregate formation in solution was determined by steady and dynamic shear rheology measurements. We observed the increased viscosity, shear thinning, and viscoelastic behavior characteristic of wormlike micelles; the rheological data are reasonably well described using a Maxwellian fluid model with a single relaxation time. In vitro release experiments demonstrated that the extension in the drug release time is dependent on the ability of a drug to form viscoelastic catanionic aggregates. Such aggregates retard the diffusive transport of drug molecules from the contact lenses. Our study revealed that the release kinetics depends on the CKC concentration and the alkyl chain length of the cationic surfactant. We demonstrated that more hydrophobic drugs such as diclofenac sodium show a more extended release than less hydrophobic drugs such as naproxen sodium.

Effect of a Cationic Surfactant on Microemulsion Globules and Drug Release from Hydrogel Contact Lenses.

The present study evaluates the in vitro release of diclofenac sodium (DFNa) from contact lenses based on poly-2-hydroxyethyl methacrylate (pHEMA) hydrogels containing an embedded microemulsion to extend release duration. The oil (ethyl butyrate)-in-water microemulsion systems are prepared with two non-ionic surfactants, Brij 97 or Tween 80, together with a long-alkyl chain cationic surfactant, cetalkonium chloride (CKC). Without CKC, Brij 97 or Tween 80-based microemulsions showed average droplet sizes of 12 nm and 18 nm, respectively. The addition of CKC decreased the average droplet sizes to 2-5 nm for both non-ionic surfactants. Such significant reduction in the average droplet size corresponds to an increase in the DFNa release duration as revealed by the in vitro experiments. Contact lens characterization showed that important properties such as optical transparency and water content of Brij 97-based contact lenses with cationic microemulsions was excellent. However, the optical transparency of the corresponding Tween 80 based contact lenses was unsatisfactory. The results indicate that cationic microemulsion-laden contact lenses can benefit from combinatory effects of microemulsions and cationic surfactant at low CKC weight percentage, e.g., with the release of 70% of the drug in 45, 10, and 7 h for B97-CKC-0.45%, CKC-0.45%, and control lenses, respectively. However, the microemulsion effect on extending DFNa release became negligible at the highest CKC weight percentage (1.8%).

Extended delivery of non-steroidal anti-inflammatory drugs through contact lenses loaded with Vitamin E and cationic surfactants.

The purpose of this study is to extend drug release from ACUVUE Oasys® and ACUVUE TruEye® silicone hydrogel contact lenses by incorporation of vitamin E in conjunction with a cationic surfactant. In ACUVUE Oasys® and ACUVUE TruEye®, the release of ketorolac tromethamine and flurbiprofen sodium is extended from hours to several days for 11% and 21% vitamin E, (weight of vitamin E / weight of dry lens) but with a considerable reduction in the amount of drug released. Cetalkonium chloride and stearylamine increased the drug loading capacity which was otherwise compromised by the addition of vitamin E in the contact lenses. In the case of diclofenac sodium, a sustained release over 150 h for both contact lenses can be achieved. It was found that the release-time-increase factor due to vitamin E has a linear dependence with the octanol-water partition coefficient of the drug in ACUVUE Oasys®. The results in this study show that contact lenses loaded with vitamin E in conjunction with cationic surfactants achieved sustained release of non-steroidal anti-inflammatory drugs (NSAIDs) within the therapeutic window.

Synthesis of silica-alginate nanoparticles and their potential application as pH-responsive drug carriers.

Composite silica-alginate nanoparticles were prepared via silica sol-gel technique using a water-in-oil microemulsion system. In our system, cyclohexane served as the bulk oil phase into which aqueous solutions of sodium alginate were dispersed as droplets that confined nanoparticle formation after addition of tetraethylorthosilicate (TEOS). Our studies showed that much of the particle growth is completed within the first 24 hours and reaction times up to 120 hours only resulted in an additional 5% increase in particle diameter. Average particle size was found to decrease with increasing water-to-surfactant molar ratio (R) and with increasing cocentration of alginate in the aqueous phase. The potential for drug loading during particle formation was demonstrated using rhodamine B as a model drug. In vitro release studies showed that particles incubated in pH 2.5 phosphate buffer released only about 7% of the drug load in 27 days, while 42% was released in pH 7.5 phosphate buffer over the same period. Analysis of the release profile suggested that rhodamine B was homogeneously distributed throughout the particle and that the drug diffusivity was 40-fold greater in pH 7.5 buffer compared to that at pH 2.5. These results suggest that silica-alginate nanoparticles could be used as a pH-responsive drug carrier for controlled drug release.

Disparities in Total Hip Arthroplasty Versus Hemiarthroplasty in the Management of Geriatric Femoral Neck Fractures.

INTRODUCTION: Recent clinical evidence suggests that total hip arthroplasty (THA) provides improved clinical outcomes as compared to hemiarthroplasty (HA) for displaced femoral neck fractures in elderly individuals. However, THA is still utilized relatively infrequently. Few studies have evaluated the factors affecting utilization and the role socioeconomics plays in THA versus HA. METHODS: In the United States, the National Inpatient Sample (NIS) database was used to identify patients treated surgically for femoral neck fracture, between 2009 and 2010. Patients were identified using International Classification of Diseases, Ninth Revision, codes for closed, transcervical femoral neck fractures and closed fractures at unspecified parts of the femoral neck. All candidate predictors of THA versus HA were entered into a multilevel mixed-effect regression model. RESULTS: Older patient age, being Asian or Pacific Islander, and having Medicaid payer status were all associated with lower odds of receiving THA. Patients with private insurance including Health Maintenance organization (HMO) had higher odds of THA as did patients with other insurance. Odds of THA were significantly lower among patients in teaching hospitals and higher at hospitals with greater THA volume. DISCUSSION: Ethnicity, payer status, hospital size, and institutional THA volume were all associated with the utilization of THA versus HA in the treatment of geriatric femoral neck fractures. LEVEL OF EVIDENCE: Level III Retrospective Cohort study.

Evaluation of Contemporary Trends in Femoral Neck Fracture Management Reveals Discrepancies in Treatment.

BACKGROUND: Recent evidence supports total hip arthroplasty (THA) as compared to hemiarthroplasty (HA) for the management of displaced femoral neck fractures in a significant subset of elderly patients. The purpose of this study was to examine trends in femoral neck fracture management over the last 12 years. METHODS: Using the National Inpatient Sample database, we identified patients treated for femoral neck fractures between 1998 and 2010 with THA, HA, or internal fixation (IF). We examined treatment trends and demographic variables including patient age, gender, socioeconomic status, and payer and hospital characteristics. RESULTS: We identified 362 127 femoral neck fracture patients treated between 1998 and 2010. Overall, there were statistically significant increases in rates of THA and HA, whereas rates of IF decreased. Total hip arthroplasty varied based on patient age, with significant increases occurring in age-groups 0 to 49 years, 50 to 59 years, 60 to 69 years, and 70 to 79 years. Utilization of THA varied significantly based on socioeconomic status and race. Patient sex, urban versus rural hospital location, and teaching versus nonteaching hospital status were not related to rates of THA. CONCLUSION: Rates of THA for femoral neck fractures increased between 1998 and 2010 in patients younger than 80 years, suggesting that surgeons are responding to clinical evidence supporting THA for the treatment of elderly femoral neck fractures. This is the first study to demonstrate this change and expose disparities in practice patterns over time in response to this evidence in the United States. Further research is indicated to explore the effect of socioeconomic status and race on femoral neck fracture management.

The Effects of Limb Elevation on Muscle Oxygen Saturation: A Near-Infrared Spectroscopy Study in Humans.

BACKGROUND: Orthopaedic and rehabilitation physicians often instruct patients to elevate a traumatized or postoperative lower extremity. Elevation is thought to improve patient comfort, as well as decrease swelling, wound complications, and the risk of compartment syndrome. Elevating a limb with increased compartment pressures, however, has been shown to reduce perfusion pressure and contribute to tissue ischemia. This investigation aims to advance our understanding of the tissue effects of limb elevation using a healthy patient model. OBJECTIVE: To quantify the effects of elevation, experimentally induced ischemia, and immobilization on muscle oxygen saturation in the human leg using near-infrared spectroscopy (NIRS). DESIGN: Experimental crossover study. SETTING: Orthopaedic Surgery research laboratory, Stanford Hospitals & Clinics. PATIENTS FOR PARTICIPATION: Twenty-six healthy volunteers. METHODS: Using transcutaneous sensors, we measured muscle oxygen saturation of the anterior compartment of the left (control) leg at 0, 15, and 30 cm of elevation relative to the heart using NIRS. A standardized short leg splint and a thigh tourniquet inflated to 50 mmHg were then applied to the right (experimental) leg to simulate a traumatized state. NIRS measurements were then repeated, again at 0, 15, and 30 cm of elevation. Muscle oxygen saturation values at various degrees of elevation of the control and experimental limb were then compared and analyzed by the use of a crossover study design and mixed-effects regression. MAIN OUTCOME MEASUREMENTS: Muscle oxygen saturation at varying levels of elevation in both the (1) control leg and (2) experimental leg in a simulated traumatic state. RESULTS: Male (18) males and female (8) patients between 22 and 62 years of age (mean 29.8 years) were enrolled. Mean regional muscle oxygen saturation (rSO2) of the control limbs at 0, 15 and, 30 cm of elevation were 74.2%, 72.5%, and 70.6%, respectively, whereas mean rSO2 of the experimental limbs were 66.3%, 65.0%, and 63.3%. A statistically significant decrease of rSO2 was observed (mean 7.65%) in the experimental limbs compared with the control limbs. As elevation increased, there was a statistically significant decrease in rSO2 of 0.12% per centimeter of elevation. Elevation did not decrease the rSO2 in the experimental limb to a greater degree than in the control limb. CONCLUSION: Increasing levels of elevation in a human limb results in progressively compromised muscle oxygen saturation as measured by NIR.

Which Fixation Device is Preferred for Surgical Treatment of Intertrochanteric Hip Fractures in the United States? A Survey of Orthopaedic Surgeons.

BACKGROUND: The best treatment for intertrochanteric hip fractures is controversial. The use of cephalomedullary nails has increased, whereas use of sliding hip screws has decreased despite the lack of evidence that cephalomedullary nails are more effective. As current orthopaedic trainees receive less exposure to sliding hip screws, this may continue to perpetuate the preferential use of cephalomedullary nails, with important implications for resident education, evidence-based best practices, and healthcare cost. QUESTIONS/PURPOSES: We asked: (1) What are the current practice patterns in surgical treatment of intertrochanteric fractures among orthopaedic surgeons? (2) Do surgical practice patterns differ based on surgeon characteristics, practice setting, and other factors? (3) What is the rationale behind these surgical practice patterns? (4) What postoperative approaches do surgeons use for intertrochanteric fractures? METHODS: A web-based survey containing 20 questions was distributed to active members of the American Academy of Orthopaedic Surgeons. Three thousand seven-hundred eighty-six of 10,321 invited surgeons participated in the survey (37%), with a 97% completion rate (3687 of 3784 responded to all questions in the survey). The survey elicited information regarding surgeon demographics, preferred management strategies, and decision-making rationale for intertrochanteric fractures. RESULTS: Surgeons use cephalomedullary nails most frequently for treatment of intertrochanteric hip fractures. Sixty-eight percent primarily use cephalomedullary nails, whereas only 19% primarily use sliding hip screws, and the remaining 13% use cephalomedullary nails and sliding hip screws with equal frequency. The cephalomedullary nail was the dominant approach regardless of experience level or practice setting. Surgeons who practiced in a nonacademic setting (71% versus 58%; p < 0.001), did not supervise residents (71% versus 61%; p < 0.001), or treated more than five intertrochanteric fractures a month (78% versus 67%; p < 0.001) were more likely to use primarily cephalomedullary nails. Of the surgeons who used only cephalomedullary nails, ease of surgical technique (58%) was cited as the primary reason, whereas surgeons who used only sliding hip screws cite familiarity (44%) and improved outcomes (37%) as their primary reasons. Of those who use only short cephalomedullary nails, ease of technique (59%) was most frequently cited. Postoperatively, 67% allow the patient to bear weight as tolerated. Nearly all respondents (99.5%) use postoperative chemical thromboprophylaxis. CONCLUSIONS: Despite that either sliding hip screw or cephalomedullary nail fixation are associated with equivalent outcomes for most intertrochanteric femur fractures, the cephalomedullary nail has emerged as the preferred construct, with the majority of surgeons believing that a cephalomedullary nail is easier to use, associated with improved outcomes, or is biomechanically superior to a sliding hip screw. The difference between what is evidence-based and what is done in clinical practice may be attributed to several factors, including financial considerations, educational experience, or inability of our current outcomes measures to reflect the experiences of surgeons. The educators, researchers, and policymakers among us must work harder to better define the roles of sliding hip screws and cephalomedullary nails and ensure that the increasing population with hip fractures receives high-quality and economically responsible care. LEVEL OF EVIDENCE: Level V, therapeutic study.

The Orthopaedic Foot and Ankle Outcomes Research (OFAR) network: feasibility of a multicenter network for patient outcomes assessment in foot and ankle.

INTRODUCTION: There is an increasing need for orthopaedic practitioners to measure and collect patient-reported outcomes data. In an effort to better understand outcomes from operative treatment, the American Orthopaedic Foot & Ankle Society (AOFAS) established the Orthopaedic Foot and Ankle Outcomes Research (OFAR) Network, a national consortium of foot and ankle orthopaedic surgeons. We hypothesized that the OFAR Network could successfully collect, aggregate, and report patient-reported outcome (PRO) data using the National Institutes of Health (NIH) Patient Reported Outcomes Measurement Information System (PROMIS). METHODS: Ten sites enrolled consecutive patients undergoing elective surgery for 1 of 6 foot/ankle disorders. Outcome instruments were collected preoperatively and at 6 months postoperatively using the PROMIS online system: Foot and Ankle Ability Measure (FAAM), Foot Function Index (FFI), and PROMIS physical function (PF) and pain computerized adaptive tests (CAT). During the 3-month period, 328 patients were enrolled; 249 (76%) had completed preoperative patient-reported outcomes data and procedure-specific data. Of these, 140 (56%) also completed 6-month postoperative patient- reported outcomes data. RESULTS: Ankle arthritis and flatfoot demonstrated consistently worse preoperative scores. Five of 6 disorders showed significant improvement at 6 months on PF CAT and FAAM, 4 of 6 showed improvement on pain interference CAT, and no disorders showed improvement on FFI. Ankle arthritis and flatfoot demonstrated the greatest magnitude of change on most patient-reported outcomes scales. CONCLUSION: We were able to enroll large numbers of patients in a short enrollment period for this preliminary study. Data were easily aggregated and analyzed. Substantial loss of follow-up data indicates a critical area requiring further effort. The AOFAS OFAR Network is undergoing expansion with goals to ultimately facilitate large, prospective multicenter studies and optimize the quality and interpretation of available outcome instruments for the foot and ankle population. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Cloud-based uniform ChIP-Seq processing tools for modENCODE and ENCODE.

BACKGROUND: Funded by the National Institutes of Health (NIH), the aim of the Model Organism ENCyclopedia of DNA Elements (modENCODE) project is to provide the biological research community with a comprehensive encyclopedia of functional genomic elements for both model organisms C. elegans (worm) and D. melanogaster (fly). With a total size of just under 10 terabytes of data collected and released to the public, one of the challenges faced by researchers is to extract biologically meaningful knowledge from this large data set. While the basic quality control, pre-processing, and analysis of the data has already been performed by members of the modENCODE consortium, many researchers will wish to reinterpret the data set using modifications and enhancements of the original protocols, or combine modENCODE data with other data sets. Unfortunately this can be a time consuming and logistically challenging proposition. RESULTS: In recognition of this challenge, the modENCODE DCC has released uniform computing resources for analyzing modENCODE data on Galaxy (https://github.com/modENCODE-DCC/Galaxy), on the public Amazon Cloud (http://aws.amazon.com), and on the private Bionimbus Cloud for genomic research (http://www.bionimbus.org). In particular, we have released Galaxy workflows for interpreting ChIP-seq data which use the same quality control (QC) and peak calling standards adopted by the modENCODE and ENCODE communities. For convenience of use, we have created Amazon and Bionimbus Cloud machine images containing Galaxy along with all the modENCODE data, software and other dependencies. CONCLUSIONS: Using these resources provides a framework for running consistent and reproducible analyses on modENCODE data, ultimately allowing researchers to use more of their time using modENCODE data, and less time moving it around.

Black sentinel lymph node and 'scary stickers'.

An unusual case is presented of a young adult patient with two black-stained, radio-nucleotide tracer-active sentinel lymph nodes biopsied following her primary cutaneous melanoma treatment. This was subsequently confirmed to be secondary to cutaneous tattoos, averting the need of an elective regional node dissection. History of tattooing and tattoo removal should therefore be obtained as a routine in all melanoma patients considered for sentinel node biopsy (SLN). SLN biopsy and any subsequent completion node dissection should be strictly staged so that proper histologic diagnosis of the sentinel node is available for correct decision making and treatment.

Immobilized thermolysin for highly efficient production of low-molecular-weight protamine--an attractive cell-penetrating peptide for macromolecular drug delivery applications.

Macromolecules present a remarkable potential as future therapeutics. However, their translation into clinical practice has been hampered by an inherently low bioavailability. Cell-penetrating peptides (CPP) have been recently shown to significantly improve on the bioavailability of macromolecules. Yet, the high cost associated with development and production of these peptides is a major factor hindering their rapid deployment beyond the laboratory. Here, we describe a facile and robust methodology for efficient and large-scale production of low-molecular-weight protamine-a potent CPP of great clinical potential. Our methodology is based on the immobilization of thermolysin, an enzyme catalyzing digestion of native protamine, on chemically surface-modified gels produced by silica sol-gel chemistry. Thermolysin was immobilized at extremely high matrix loading of 733 mg/g matrix and exhibited good thermal and pH stability, indicating robustness with respect to processing conditions. The mechanical properties of the silica matrix further allowed utilization of the immobilized thermolysin in both batch and packed-bed reactor systems to produce the LMWP peptide in high yields. Results presented here are of high significance as this efficient and cost-effective production of high purity LMWP could enable clinical translation of many potential macromolecular drugs.