Immobilization Protocol Research Articles

Turn-on Fluorescence Hg<sup>2+ </sup>Chemosensing Based in a Rhodamine 6G Derivative and Different Sensing Immobilization Approaches

In this study, the advantages and disadvantages of different immobilization approaches, proposed for a luminescent Hg2+chemosensor based in a spirocyclic phenyl-thiosemicarbazide Rhodamine 6G derivative (FC1), are analysed, and a comparative study of the different immobilization protocols is performed. The results demonstrated that the chemosensor is able to detect Hg2+ at ng mL-1 trace levels, with a remarkable selectivity, allowing its determination in biological, toxicological and environmental samples. Methyl methacrylate (MMA) and hydroxyethylmethacrylate (HEMA) polymeric film, electrospinning generated polymeric microfibres, nylon membranes and Au nanoparticles were tested as immobilization materials. The utilization of gold nanoparticles (AuNPs) is the method with the lowest limit of detection (LOD = 0.15 ng mL-1), but the response time is too high for practical use (7 hours). Nylon membranes give a very low detection limit (0.4 ng mL-1) and a practical response time (4 min), being the method of choice for practical applications to determine Hg2+ in aquatic, biological and toxicological samples, at a sampling rate of about 15 samples per hour.

Rehabilitation Protocols After Basal Joint Surgery: All over the Map: Commentary on an article by Douglas T. Hutchinson, MD, et al.: "A Prospective, Randomized Trial of Mobilization Protocols Following Ligament Reconstruction and Tendon Interposition".

Commentary Hutchinson et al. have designed a carefully done, Level-II study that compares 2 different postoperative immobilization protocols following ligament reconstruction and tendon interposition. They found that 4 weeks of thumb-spica splint immobilization followed by 4 weeks of intermittent splinting (early mobilization group) provided outcomes that were virtually comparable with those provided by 6 weeks of immobilization with use of a forearm-based thumb-spica splint followed by 6 weeks of intermittent splinting (immobilization group). Much of what is taught and done in musculoskeletal medicine is based on what we have learned from our mentors or what we have gleaned from the literature. Unfortunately, the literature frequently falls short and therapeutic decisions are based on Level-IV or Level-V evidence. As Hageman et al. pointed out, when evidence is inconclusive, surgeons default to “works in my hands,” “familiarity with treatment,” and “what my mentor taught me.”1 To address this situation and better serve our patients, the American Academy of Orthopaedic Surgeons and many specialty societies have worked diligently to publish Clinical Practice Guidelines (CPGs) and Appropriate Use Criteria (AUC). The CPGs are based on the studies with the highest levels of evidence available in the peer-reviewed literature; unfortunately, much of the evidence is inconclusive, and hence there is the need for more Level-I and Level-II studies. AUC combine expert opinion with the findings of studies with the best levels of evidence in order to guide practitioners on when it is appropriate to perform a procedure under a specific set of circumstances. Regrettably, there are no CPGs or AUC for many of the procedures that surgeons commonly perform or for those that have, to date, not gained a great deal of traction. In one of the earliest articles about basal joint arthroplasty, Gervis reported on 15 patients who underwent trapeziectomy alone2. Only 2 patients had “inferior” results. The postoperative recommendations in the study were brief: initiate an active range of motion “at once” and use “supervision” as necessary to achieve a full range of motion. Since then, in a systematic review, Wolfe et al. pointed out that rehabilitation protocols have become more detailed but recommendations are all over the map3. The focus of most of the literature on basal joint arthritis is on surgical technique. When developing a rehabilitation program following basal joint arthroplasty, it is key to consider the length of time for complete thumb immobilization, when to initiate an active range of motion, and the total time that the splint should be worn because these factors affect cost, quality of life while recuperating, and return to full activity. In the study by Hutchinson et al., 2 secondary considerations were noted. The type of splint (forearm-based versus hand-based) did not seem to matter. The treatment in the immobilization group was supplemented by 6 weeks with a forearm-based splint whereas the early mobilization group utilized a less restrictive hand-based splint for 4 weeks. On a similar note, wrist and thumb strengthening was initiated at 8 weeks in the early mobilization group and 12 weeks in the immobilization group. Again, outcome was not affected. I was surprised to learn that simultaneous procedures—particularly those on the metacarpophalangeal joint—had no effect on outcome when comparing the 2 groups. Finally, an aspect not addressed in this study is the role of supervised hand therapy and how it affects outcome. In their systematic review, Wolfe et al. noted that some authors recommended no formal therapy whereas others endorsed a very structured rehabilitation program3.

Comparison of two protocols for field immobilization of white-eared opossums (Didelphis albiventris)

Fil: Waxman Dova, Samanta. Universidad de Buenos Aires. Facultad de Cs.veterinarias. Catedra de Anestesiologia y Algiologia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina

Chemical immobilization protocols in free‐ranging South American fur seal (Arctocephalus australis) and adult female South American sea lion (Otaria byronia)

Chemical immobilization protocols in free‐ranging South American fur seal (<i>Arctocephalus australis</i>) and adult female South American sea lion (<i>Otaria byronia</i>)

One Brace: One Visit: Treatment of Pediatric Distal Radius Buckle Fractures With a Removable Wrist Brace and No Follow-up Visit.

Previous studies have showed the efficacy of removable brace treatment for distal radius buckle fractures in children, whereas others have independently suggested that these injuries do not require additional radiographic imaging. However, no study has sought to collectively determine whether treating pediatric distal radius buckle fractures with a removable brace and no follow-up visit or imaging after the initial visit is a safe and satisfactory protocol. In total, 42 consecutive patients with a distal forearm buckle fracture seen by a single fellowship trained pediatric orthopaedic surgeon were eligible to participate. Two patients refused participation, yielding 40 patients treated with a standard protocol of immobilization with a removable wrist brace for a prescribed period of time with no additional imaging or clinical follow-up. Two staggered telephone surveys were then conducted. The first survey was conducted within 1 week of the designated brace-removal date to determine the exact date the brace was discontinued. The second survey was conducted 5 to10 months postinjury to determine patient outcomes and parent satisfaction. In total, 100% of patients were reached for the initial survey and 90% (36/40) of patients were reached for the secondary survey. There were no complications, including refracture or residual pain, following treatment. In total, 100% of parents felt their child had returned to full and normal function and all said they would choose to have the same treatment again. In total, 67% of parents would have had to take time off from work and 77% of children would have missed school if they had hypothetically been required to attend a follow-up appointment. Treatment of pediatric distal forearm buckle fractures with a removable wrist brace and no follow-up visit or radiographs results in both excellent patient outcomes and parental satisfaction. Level IV-case series.

Ligand Binding Stabilizes Cellulosomal Cohesins as Revealed by AFM-based Single-Molecule Force Spectroscopy

The cohesin-dockerin receptor-ligand family is the key element in the formation of multi-enzyme lignocellulose-digesting extracellular complexes called cellulosomes. Changes in a receptor protein upon binding of a ligand - commonly referred to as allostery - are not just essential for signalling, but may also alter the overall mechanical stability of a protein receptor. Here, we measured the change in mechanical stability of a library of cohesin receptor domains upon binding of their dockerin ligands in a multiplexed atomic force microscopy-based single-molecule force spectroscopy experiment. A parallelized, cell-free protein expression and immobilization protocol enables rapid mechanical phenotyping of an entire library of constructs with a single cantilever and thus ensures high throughput and precision. Our results show that dockerin binding increases the mechanical stability of every probed cohesin independently of its original folding strength. Furthermore, our results indicate that certain cohesins undergo a transition from a multitude of different folds or unfolding pathways to a single stable fold upon binding their ligand.

Modulation of the Catalytic Properties of Lipase B from Candida antarctica by Immobilization on Tailor-Made Magnetic Iron Oxide Nanoparticles: The Key Role of Nanocarrier Surface Engineering.

The immobilization of biocatalysts on magnetic nanomaterial surface is a very attractive alternative to achieve enzyme nanoderivatives with highly improved properties. The combination between the careful tailoring of nanocarrier surfaces and the site-specific chemical modification of biomacromolecules is a crucial parameter to finely modulate the catalytic behavior of the biocatalyst. In this work, a useful strategy to immobilize chemically aminated lipase B from Candida antarctica on magnetic iron oxide nanoparticles (IONPs) by covalent multipoint attachment or hydrophobic physical adsorption upon previous tailored engineering of nanocarriers with poly-carboxylic groups (citric acid or succinic anhydride, CALBEDA@CA-NPs and CALBEDA@SA-NPs respectively) or hydrophobic layer (oleic acid, CALBEDA@OA-NPs) is described. After full characterization, the nanocatalysts have been assessed in the enantioselective kinetic resolution of racemic methyl mandelate. Depending on the immobilization strategy, each enzymatic nanoderivative permitted to selectively improve a specific property of the biocatalyst. In general, all the immobilization protocols permitted loading from good to high lipase amount (149 < immobilized lipase < 234 mg/gFe). The hydrophobic CALBEDA@OA-NPs was the most active nanocatalyst, whereas the covalent CALBEDA@CA-NPs and CALBEDA@SA-NPs were revealed to be the most thermostable and also the most enantioselective ones in the kinetic resolution reaction (almost 90% ee R-enantiomer). A strategy to maintain all these properties in long-time storage (up to 1 month) by freeze-drying was also optimized. Therefore, the nanocarrier surface engineering is demonstrated to be a key-parameter in the design and preparation of lipase libraries with enhanced catalytic properties.

A novel approach for bioconjugation of Rhizomucor miehei lipase (RML) onto amine-functionalized supports; Application for enantioselective resolution of rac-ibuprofen

Since the amine groups are highly reactive toward various functional moieties, the formation of covalent bonds between a biomolecule and an insoluble amine-functionalized support is the most frequently used technique in immobilization protocols. A new approach based on the Ugi four-component reaction was used for immobilization of Rhizomucor miehei lipase (RML) as a model enzyme on amine-functionalized silica and silica nanoparticles (SBA-15). For this, the amine-modified supports were prepared and the structural properties of the functionalized supports, prior to and after functionalization were characterized by using IR, SEM, TGA, DTA, TEM. Immobilization of RML on the aminated carriers was performed under extremely mild conditions (25 °C, pH 7).The results revealed very rapid immobilization of 150 and 200 mg of RML on 1 g of silica-NH2 and SBA-NH2, respectively, producing 95–100% of immobilization yield. The specific activity and optimum pH of the immobilized preparations and the effect of temperature and co-solvents on their stabilities as well as the reusability of the derivatives were tested. The immobilized preparations were also used as enantioselective catalyst in kinetic resolution of racemic ibuprofen. Among them, Silica-RML showed the best selectivity with 92.2% enantiomeric excess (ee) and E-value of 33.9.

Kinetic resolution of drug intermediates catalyzed by lipase B from Candida antarctica immobilized on immobead-350.

Novozyme 435, which is a commercial immobilized lipase B from Candida antarctica (CALB), has been proven to be inadequate for the kinetic resolution of rac-indanyl acetate. As it has been previously described that different immobilization protocols may greatly alter lipase features, in this work, CALB was covalently immobilized on epoxy Immobead-350 (IB-350) and on glyoxyl-agarose to ascertain if better kinetic resolution would result. Afterwards, all CALB biocatalysts were utilized in the hydrolytic resolution of rac-indanyl acetate and rac-(chloromethyl)-2-(o-methoxyphenoxy) ethyl acetate. After optimization of the immobilization protocol on IB-350, its loading capacity was 150 mg protein/g dried support. Furthermore, the CALB-IB-350 thermal and solvent stabilities were higher than that of the soluble enzyme (e.g., by a 14-fold factor at pH 5-70°C and by a 11-fold factor in dioxane 30%-65°C) and that of the glyoxyl-agarose-CALB (e.g., by a 12-fold factor at pH 10-50°C and by a 21-fold factor in dioxane 30%-65°C). The CALB-IB-350 preparation (with 98% immobilization yield and activity versus p-nitrophenyl butyrate of 6.26 ± 0.2 U/g) was used in the hydrolysis of rac-indanyl acetate using a biocatalyst/substrate ratio of 2:1 and a pH value of 7.0 at 30°C for 24 h. The conversion obtained was 48% and the enantiomeric excess of the product (e.e.p ) was 97%. These values were much higher than the ones obtained with Novozyme 435, 13% and 26% of conversion and e.e.p, respectively. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:878-889, 2018.

Detection of heart-type fatty acid-binding protein (h-FABP) using piezoresistive polymer microcantilevers functionalized by a dry method

Piezoresistive microcantilever-based sensor platform is being used for the last two decades due to their low cost, rapid response and label-free detection system. In this work, we are reporting a microfabricated piezoresistive SU-8/carbon black (polymer cantilever)-based sensor platform for the detection of a clinically important early-stage cardiac marker, i.e., fatty acid-binding protein. It is a most preferred cardiac marker for the diagnosis of acute myocardial infarction. The embodiment of the sensor is a SU-8 microcantilever chip with an integrated nanoparticle composite (carbon black) as a piezoresistor for on-chip electrical transduction. Prior to improving the sensing and susceptibility towards the specific target biomolecule (i.e., h-FABP), the fabricated SU-8 polymer cantilevers were subjected to tailored functionalization. This includes the use of an in-house dry method of hot wire chemical vapour deposition technique to graft amine groups onto the SU-8 surface. The surface-modified microcantilevers were further integrated with a polydimethylsiloxane liquid flow cell and connected externally with an electrical read-out system. Immobilization of the antibody corresponding to the marker protein on the microcantilever surface and subsequent recording of the signal generated upon the antibody–antigen interaction were carried out inside the liquid flow cell. Using our optimized immobilization protocol with this experimental set-up, we were successfully able to detect h-FABP concentration as low as 100 ng/ml.

Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody

BackgroundG-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens. MethodsSPR was employed to characterize the anti-G4 antibody interaction with G4 and non-G4 oligonucleotides. Dextran-functionalized sensor chips were used both in covalent coupling and capturing procedures. ResultsThe use of two leading molecule for orienting the antibody of interest allowed to improve its activity from completely non-functional to 65% active. The specificity of the anti-G4 antobody for G4 structures could thus be assessed with high sensitivity and reliability. ConclusionsOptimization of the immobilization protocol for SPR biosensing, allowed us to determine the anti-G4 antibody affinity and specificity for G4 antigens with higher sensitivity with respect to other in vitro assays such as ELISA. Anti-G4 antibody specificity is a fundamental assumption for the future utilization of this kind of antibodies for monitoring G4s directly in cells. General significanceThe heterogeneous orientation of amine-coupling immobilized ligands is a general problem that often leads to partial or complete inactivation of the molecules. Here we describe a new strategy for improving ligand orientation: driving it from two sides. This principle can be virtually applied to every molecule that loses its activity or is poorly immobilized after standard coupling to the SPR chip surface.

Displaced midshaft clavicle fracture in athletes – should we operate?

ObjectiveTo assess the results of the treatment of fractures of the middle third of the clavicle with a pre-contoured plate in athletes. MethodsThe authors performed 26 osteosyntheseis in 25 patients with fractures of the middle third of the clavicle. The fractures were classified according to Robinson as 16 type B1 and ten type B2. The mean age was 37 years, ranging from 15 to 63 years, 20 patients were male and five were female, and all play sports in an amateur or professional manner. All patients were operated on in the acute phase of fracture within five days after trauma. Patients were treated with open reduction and internal fixation using pre-contoured plates. The technique and the implant used were the same, but the time of immobilization and rehabilitation protocol were individualized for each patient according to physical activity. Mean follow-up was 16.8 months (6–48 months), with return to sport activities in 45.6 days. ResultsFunctional assessment was performed using the University of California at Los Angeles (UCLA) score. The results averaged 34.07 of 35 points. No cases presented nonunion. Only one complication was observed (thrombosis of the subclavian vein with good response to conservative treatment); two patients required implant removal. ConclusionThe surgical treatment of clavicle fractures in athletes presented satisfactory functional outcome and early return sport.

Compulsory Use of the Backboard is Associated with Increased Frequency of Thoracolumbar Imaging

Background: Backboards have been shown to cause pain in uninjured patients. This may alter physical exam findings, leading emergency department (ED) providers to suspect a spinal injury when none exists resulting in additional imaging of the thoracolumbar spine. New York had previously employed a “Spinal Immobilization” protocol that included compulsory backboard application for all patients with suspected spinal injuries. In 2015, New York instituted a new “Spinal Motion Restriction” protocol that made backboard use optional for these patients. The objective of this study was to determine if this protocol change was associated with decreased backboard utilization and ED thoracolumbar spine imaging. Methods: This was a retrospective before-and-after chart review of subjects transported by a single emergency medical services (EMS) agency to one of four EDs for emergency calls dispatched as motor vehicle collisions (MVC). EMS and ED data were included for all calls within a 6-month interval before and after the protocol change. The protocol change was implemented in the second half of 2015. Subject demographics, backboard use, and spine imaging were reviewed for the intervals January–June 2015 and January–June 2016. Results: There were 818 subjects in the before period and 796 subjects in the after period. Subjects were similar in terms of gender, age and type of MVC in both periods. A backboard was utilized for 440 (54%) subjects in the before period and 92 (12%) subjects in the after period (p < 0.001). ED thoracic spine imaging was performed on 285 (35%) subjects in the before period, and 235 (30%) subjects in the after period (p = 0.02). ED lumbar spine imaging was performed for 335 (41%) subjects in the before period, and 281 (35%) subjects in the after period (p = 0.02). Conclusion: A shift from a spinal immobilization protocol to a spinal motion restriction protocol was associated with a decrease in backboard utilization by EMS providers and a decrease in thoracolumbar spine imaging by ED providers.

In-flow protein immobilization monitored by magnetic resonance imaging

Protein immobilization is a key enabling technology for flow biocatalysis. For this purpose, many different immobilization protocols and characterization techniques have been developed in recent decades. However, examples where proteins are directly immobilized on ready-to-use reactors are scarce, likely due to the lack of analytical tools to monitor in-flow protein immobilization in a non-invasive manner. Here, we have for the first time exploited Magnetic Resonance Imaging (MRI) to characterize in-flow protein immobilization on pre-packed bed columns. This concept was demonstrated by in-flow immobilization of a green fluorescence protein. MRI analysis revealed that both the protein concentration of the flushed solution and flow rate play key roles in controlling the spatial organization of the protein across the packed-bed reactor. This analytical tool coupled to in-flow protein immobilization has been expanded to more industrially relevant enzymes, such as the lipase from Thermomyces lanuginosus, achieving a ready-to-use reactor packed with a heterogeneous biocatalyst with high activity (up to 3000 U × g−1) and high stability (75% residual activity after 1 h incubation at 60 °C). Introducing new analytical tools during the fabrication of heterogeneous biocatalysts will contribute to make the process of immobilizing proteins on solid carriers more rational than currently is.

New Immobilization Guidelines Change EMS Critical Thinking in Older Adults With Spine Trauma

Objective: The impact of immobilization techniques on older adult trauma patients with spinal injury has rarely been studied. Our advisory group implemented a change in the immobilization protocol used by emergency medical services (EMS) professionals across a region encompassing 9 trauma centers and 24 EMS agencies in a Rocky Mountain state using a decentralized process on July 1, 2014. We sought to determine whether implementing the protocol would alter immobilization methods and affect patient outcomes among adults ≥60 years with a cervical spine injury. Methods: This was a 4-year retrospective study of patients ≥60 years with a cervical spine injury (fracture or cord). Immobilization techniques used by EMS professionals, patient demographics, injury characteristics, and in-hospital outcomes were compared before (1/1/12–6/30/14) and after (7/1/14–12/31/15) implementation of the Spinal Precautions Protocol using bivariate and multivariate analyses. Results: Of 15,063 adult trauma patients admitted to nine trauma centers, 7,737 (51%) were ≥60 years. Of those, 237 patients had cervical spine injury and were included in the study; 123 (51.9%) and 114 (48.1%) were transported before and after protocol implementation, respectively. There was a significant shift in the immobilization methods used after protocol implementation, with less full immobilization (59.4% to 28.1%, p < 0.001) and an increase in the use of both a cervical collar only (8.9% to 27.2%, p < 0.001) and not using any immobilization device (15.5% to 31.6%, p = 0.003) after protocol implementation. While the proportion of patients who only received a cervical collar increased after implementing the Spinal Precautions Protocol, the overall proportion of patients who received a cervical collar alone or in combination with other immobilization techniques decreased (72.4% to 56.1%, p = 0.01). The presence of a neurological deficit (6.5% vs. 5.3, p = 0.69) was similar before and after protocol implementation; in-hospital mortality (adjusted odds ratio = 0.56, 95% confidence interval: 0.24–1.30, p = 0.18) was similar post–protocol implementation after adjusting for injury severity. Conclusions: There were no differences in neurologic deficit or patient disposition in the older adult patient with cervical spine trauma despite changes in spinal restriction protocols and resulting differences in immobilization devices.

Fungal synthesis of chiral phosphonic synthetic platform – Scope and limitations of the method

Chiral hydroxyphosphonates due to their wide range of biological properties are industrially important chemicals. Chemical synthesis of their optical isomers is expensive, time consuming and not friendly to the environment, so biotransformations are under consideration. Among others, these compounds act as enzymes inhibitors. This makes the bioconversions of phosphonates, especially scaling experiments, hard to perform. Biocatalysis is one of the methods that can be applied in synthesis of optically pure compounds. To increase the efficiency of the process with whole cell biocatalysts, it is essential to ensure optimal reaction conditions that minimize cellular stress and can enhance the metabolic activity of cells. The present investigation focuses on the scaling up of the kinetic resolution of racemic mixture of 2-butyryloxy-2-(ethoxy-P-phenylphosphinyl)acetic acid, applying free and immobilized form of the fungal biocatalysts and two operation systems: shake flask and recirculated fixed-bed batch reactor. Protocols of effective mycelium immobilization on polyurethane foams were set for T. purpurogenus IAFB 2512, F. oxysporum, P. commune. The best results of biotransformation were obtained with the immobilized P. commune in the column recirculated fixed-bed batch reactor. The conversion reaches 56% (maximal for the kinetic process) and the enantiomeric enrichment of the isomers mixture ranges between 82 and 93% (93% for ester of RP,R conformation). All biocatalysts exhibit SP-preference toward tested compound, what is essential because of importance of the phosphorus atom chirality for its biological activity.

Do demographic and perioperative parameters really affect the final outcomes of pediatric femur shaft fractures managed by elastic nails? A prospective study

ObjectivePediatric femoral shaft fractures are more commonly treated with intramedullary titanium elastic nail system (TENS). Adhering to the principles, most studies had supported excellent results with this instrumentation and attributed the variation in age, weight, immobilization protocols, technical factors like fracture pattern, reduction and complications as reasons to poor outcomes in their individual studies. Hence, we wanted to identify the potential demographic and perioperative parameters that could affect the final outcomes in this cohort. MethodsA prospective (level III) study done in a single center between November 2013 and January 2017 on isolated closed femoral shaft fractures in patients of age between 6 and 15 years managed with TENS. The demographic and perioperative parameters were recorded. The patients were followed up regularly with plain radiographs. The final outcomes were computed at end of one year and recorded as poor, satisfactory and excellent as defined by Flynn criteria. ResultsAmong the thirty patients included, mean age was 8.2 years. 80% of the patients weighing over 40 kg had satisfactory to poor outcomes. (p = 0.005). 45% of patients with proximal and distal level fractures that were long oblique spiral or comminuted types had satisfactory outcomes; however it was not statistically significant. The mean delay to surgical fixation was 5.87 days, surgical time was between 45 and 150 min and open reduction was required in about 17 cases (57%). Patients with immobilization beyond 6 weeks had satisfactory outcomes (p = 0.001). We had 5 patients with minor complications (4 bursitis and 3 superficial infections) and one major complication (chronic osteomyelitis and deep venous thrombosis) leading to satisfactory and poor outcomes respectively. Significant osseous union was noted between 6 and 11 months with 97% of patients attaining union within 9 months. ConclusionsIntramedullary TENS is an excellent modality to treat femoral shaft fractures in patients of school going age. However, factors like weight of the patient >40 kg, immobilization beyond 6 weeks, minor and major complications in the perioperative period could pose risks for poor to satisfactory outcomes and should be anticipated and explained accordingly.

Signal Enhancement of SPR Biosensor Detection by Using of Gold Nanoparticles for Human beta-2-Microglobulin as a Model Protein Biomarker

The highly sensitive method of surface plasmon resonance (SPR) detection of low concentrations of target proteins based on the biosensor signal enhancement by using gold nanoparticles (similar to “sandwich” assay type) is described. The commercial protein preparations of beta-2-microglobulin (B2M) as a model biomarker and polyclonal (Pab) and monoclonal antibodies (Mab) to B2M were used. It has been shown that this universal and reproducible method can be applied for SPR analysis of other protein biomarkers by analogy with the biomarker protein B2M. The present work is also focused on the experimental protocol description. The protocols of gold nanoparticles (GNP) synthesis, obtaining the conjugates of Pab/GNP and measuring their concentration, the protocol of Mab covalent immobilization on the optical chip CM5 of a biosensor and also SPR registration of molecular interactions Mab-biomarker and in the “sandwich” assay type Mab-biomarker-Pab or Mab-biomarker-Pab/GNP are considered in detail.

A Study of the Oscillatory Components of the Skin Microhemodynamics in Mice by Laser Doppler Flowmetry

A skin blood flow was studied by laser doppler flowmetry. Fourteen male BALB/c mice were used. The skin blood flow was registered in the left hind paw. To measure the skin microhemodynamics, we proposed a protocol of animal immobilization with two types of anesthesia, injection with zoletil and inhalation with nitrous oxide. The borders of low-frequency intervals of oscillations of the microhemodynamics in mice have been determined; they coincided with those for humans and rats that were determined earlier.

Immobilization and image-guidance methods for radiation therapy of limb extremity soft tissue sarcomas: Results of a multi-institutional survey

Radiation therapy for limb-extremity soft tissue sarcoma (STS) requires accurate, reproducible dose delivery. However, patient positioning is challenging and there is a lack of existing guidelines to assist institutional standardization. Therefore, we conducted a multi-institutional international survey of STS immobilization, image guidance methods, and treatment protocols to investigate current practice. Seventy-three UK radiotherapy centers and 15 hospitals in 7 other countries completed a questionnaire on STS immobilization and image-guidance procedures. Specifically, the survey collated information on the current usage of immobilization equipment, including custom devices, patient setup tolerances, the use of written protocols, the modality and frequency of image guidance, the method of treatment, allocated treatment times, and the application of surgical clips. Multiple combinations of immobilization devices were reported. In the UK, 12%, 40%, 30%, 12%, and 5% use 1, 2, 3, 4, and 5 types of device for lower limb STS. Vacuum bag plus either foot or ankle support was most common (66%). Of 15 international centers, 27%, 60%, 7%, 0%, 7% use 1, 2, 3, 4, 5 devices, with vacuum bags (73%) and thermoplastic (47%) predominant, similar to UK values of 77% and 52%. For image guidance, in the UK, 37% use kV planar, 34% use MV planar, and 16% use cone-beam CT for the first 3 fractions and then weekly. Internationally, daily imaging was more prevalent with 33% using kV planar, 7% MV planar, and 40% cone-beam CT daily. Custom devices plus combinations of devices, along with 5- and 10-mm set-up tolerances, were most commonly reported. Less than half of centers have written treatment protocols. Conventional treatment is most common in the UK, with only 42% using conformal techniques. Treatment is allocated between 10 and 30 minutes. Twenty-six percent of UK centers and 53% of international centers use surgical clips. Across treatment centers, there is no consistent approach to STS immobilization, image-guidance methods, or treatment protocols assessed by this survey. A wide variety of immobilization devices and configurations are utilized, and the frequency and modality of imaging are similarly diverse. In the absence of guidelines, the creation of an online repository of example immobilization techniques could enable centers to compare a diversity of cases. The availability of a forum for viewing and discussing a range of cases could potentially lead to improved patient setup and reduce the time taken to devise an individual immobilization strategy.