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Mikecoxite, ideally (CHg4)OCl2, is the first mercury-oxide-chloride-carbide containing a C4– anion coordinated by four Hg atoms (a permercurated methane derivative) to be described as a mineral species. It was found at the McDermitt open-pit mine on the eastern margin of the McDermitt Caldera, Humboldt County, Nevada, U.S.A. It is monoclinic, space group P21/n, Z = 4; a = 10.164(5), b = 10.490(4), c = 6.547(3) Å, V 698.0(5) Å3. Chemical analysis by electron microprobe gave Hg 86.38, Cl 11.58, Br 0.46, C 1.81, sum = 100.23 wt%, and O was detected but the signal was too weak for quantitative chemical analysis. The empirical formula, calculated on the basis of Hg + Cl + Br = 6 apfu, is (C1.19Hg3.39)(Cl2.57Br0.05)Σ2.62, and the ideal formula based on the chemical analysis and the crystal structure is (CHg4)OCl2. The seven strongest lines in the X-ray powder diffraction pattern are [d (Å), I, (hkl)]: 2.884, 100, (230); 2.989, 81, (301, 301, 112, 112, 131, 131); 2.673, 79, (122, 122, 212, 212); 1.7443, 40, (060, 432, 432); 5.49, 34, (101, 101); 4.65, 32, (120); 2.300, 30, (312, 312). The Raman spectrum shows three bands at 638, 675, and 704 cm–1, well above the range characteristic of NHg4 stretching vibrations between 540 and 580 cm–1, that are assigned to CHg4 stretching vibrations. Mikecoxite forms intergrowths of bladed crystals up to 100 μm long that occur on granular quartz or in vugs associated with kleinite. It is black with a submetallic to metallic luster and strong specular reflections and does not fluoresce under short-or long-wave ultraviolet light. Neither cleavage nor parting were observed, and the calculated density is 8.58 g/cm3. In the crystal structure of mikecoxite, (C4–Hg42+) groups link through O2– ions to form three-membered rings that polymerize into corrugated [CHg4OCl]+ layers with near-linear C4––Hg2+–O and C 4––Hg2+–Cl linkages. The layers link in the third direction directly via weak Hg2+–O2– and Hg2+–Cl– bonds to adjacent layers and also indirectly via interlayer Cl–. A bond-valence parameter has been derived for (Hg2+–C4–) bonds: Ro = 2.073 Å, b = 0.37, which gives bond-valence sums at the C4– ions in accord with the valence-sum rule. The source of carbon for mikecoxite in the volcanic high-desert environment of the type locality seems to be methane, with the reaction catalyzed by microbiota through full mercuration of carbon atoms, beyond the first stage that produces the volatile and highly mobile methylmercury, [CH3Hg]+, a potent neurotoxin that accumulates in marine food chains. Both the mineral and the mineral name have been approved by the Commission on New Minerals, Nomenclature and Classification of the International Mineralogical Association (IMA 2021-060). The mineral is named after Michael F. Cox (b. 1958), a founding member of the New Almaden Quicksilver County Park Association (NAQCPA) who was responsible for characterizing and remediating environmental mercury on-site and who recovered the rock containing the new mineral.

Although everything seemed clear about the Ag-Sb-S compounds belonging to one of the more deeply studied experimental systems, nature allowed us to discover a new polymorph of Ag3SbS3, which could represent a compound for assessing new technological potentialities. The new mineral species pyradoketosite, Ag3SbS3 (IMA 2019-132), was discovered in the pyrite + baryte + iron oxide ore deposit of the Monte Arsiccio mine, Apuan Alps, Tuscany, Italy. It occurs as brittle orange acicular crystals, up to 200 μm in length and 25 μm in thickness, with adamantine luster. Under reflected light, pyradoketosite is slightly bluish-gray, with abundant orange internal reflections. Bireflectance is weak, and anisotropism was not observed, being masked by abundant internal reflections. Minimum and maximum reflectance data for the wavelengths recommended by the Commission on Ore Mineralogy [Rmin/Rmax (%) (λ, nm)] are 32.8/32.9 (470), 30.2/30.7 (546), 29.0/29.6 (589), and 27.5/28.4 (650). Electron microprobe analysis gave (mean of 6 spot analyses, in wt%): Ag 59.81, Sb 22.63, S 17.78, total 100.22. On the basis of (Ag+Sb) = 4 atoms per formula unit, the empirical formula of pyradoketosite is Ag2.996(11)Sb1.004(11)S2.996(15). Pyradoketosite is monoclinic, space group P21/n, with a = 13.7510(15), b = 6.9350(6), c = 19.555(2) Å, β = 94.807(4)°, V = 1858.3(3) Å3, Z = 12. The crystal structure was solved and refined to R1 = 0.063 on the basis of 2682 unique reflections with Fo > 4σ(Fo) and 191 refined parameters. The structure of pyradoketosite can be described as formed by the alternation of {101} layers: an Sb-rich layer, Sb3AgS3, and two distinct Ag8S6 layers. This layered organization allows identifying structural relationships with the wittichenite-skinnerite pair. Pyradoketosite is associated with pyrargyrite, tetrahedrite-(Hg), valentinite, and probable pyrostilpnite in baryte + dolomite + quartz veins embedded in metadolostone. Its name derives from the old Greek words “πυρ” (fire) and “άδόκητος” (unforeseen), because of the unexpected occurrence of this third polymorph of the compound Ag3SbS3.

Progressive supranuclear palsy (PSP) is a neurodegenerative condition, typically presenting with, but not limited to, impairments of postural instability, gait, and gaze stability.This case report describes the multifactorial assessment and rehabilitation of a patient with atypical PSP who has significant gaze deficits, asymmetrical stepping responses, trunk rigidity, and reduced posterior excursion on limits of stability.Evaluation utilized computerized gait and balance assessments, foot clearance analysis, a squat test, and a timed stepping test. The intervention included boxing, stepping tasks, and treadmill training each with eye movement challenges. A total of 15 hours of physical therapy was provided; 1 hour, 2 times a week.Post-intervention improvements were noted subjectively, on eye-body coordination, and objectively, on limits of stability, foot clearance, and task performance (squats, timed stepping). Follow-up demonstrated some decline from posttest results; however, patient-reported adherence to the program was less than recommended.A multifactorial rehabilitation program can improve balance, eye-body coordination, and strength in a high functioning patient with atypical PSP. Longitudinal randomized controlled studies are suggested to further investigate this interventional approach in high functioning individuals diagnosed with atypical PSP.

Background A cost effective and efficient diagnostic tool for COVID-19 as near to the point of care (PoC) as possible would be a game changer in the current pandemic. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 min, alongside standard methods in a real-life clinical setting. Methods This prospective service improvement project piloted an RT-LAMP method on nasal and pharyngeal swabs on 21 residents of a high dependency care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of a single swab using RT-LAMP compared with the current standard, RT-PCR, as per Standards for Reporting Diagnostic Accuracy Studies (STARD) guidelines. Results The novel method accurately detected 8/10 RT-PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a “gold standard”, the sensitivity and specificity of a single novel test were 80 and 73% respectively. PPV was 73% and NPV was 83%. Incorporating retesting of low signal RT-LAMP positives improved the specificity to 100%. We also speculate that hypothermia may be a significant early clinical sign of COVID-19. Conclusions RT-LAMP testing for SARS-CoV-2 was found to be promising, fast and to work equivalently to RT-PCR methods. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the PoC. RT-LAMP could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread.

Background Accurate source localization from electroencephalography (EEG) requires electrode co-registration to brain anatomy, a process that depends on precise measurement of 3D scalp locations. Stylus digitizers and camera-based scanners for such measurements require the subject to remain still and therefore are not ideal for young children or those with movement disorders. New method Motion capture accurately measures electrode position in one frame but marker placement adds significant setup time, particularly in high-density EEG. We developed an algorithm, named MoLo and implemented as an open-source MATLAB toolbox, to compute 3D electrode coordinates from a subset of positions measured in motion capture using spline interpolation. Algorithm accuracy was evaluated across 5 different-sized head models. Results MoLo interpolation reduced setup time by approximately 10 min for 64-channel EEG. Mean electrode interpolation error was 2.95 ± 1.3 mm (range: 0.38–7.98 mm). Source localization errors with interpolated compared to true electrode locations were below 1 mm and 0.1 mm in 75 % and 35 % of dipoles, respectively. Comparison with existing methods MoLo location accuracy is comparable to stylus digitizers and camera-scanners, common in clinical research. The MoLo algorithm could be deployed with other tools beyond motion capture, e.g., a stylus, to extract high-density EEG electrode locations from a subset of measured positions. The algorithm is particularly useful for research involving young children and others who cannot remain still for extended time periods. Conclusions Electrode position and source localization errors with MoLo are similar to other modalities supporting its use to measure high-density EEG electrode positions in research and clinical settings.

BackgroundThe need for a fast and reliable test for COVID-19 is paramount in managing the current pandemic. A cost effective and efficient diagnostic tool as near to the point of care (PoC) as possible would be a game changer in current testing. We tested reverse transcription loop mediated isothermal amplification (RT-LAMP), a method which can produce results in under 30 minutes, alongside standard methods in a real-life clinical setting.MethodsThis service improvement project piloted a research RT-LAMP method on nasal and pharyngeal swabs on 21 residents in a high dependency care home, with two index COVID-19 cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (RT-PCR). We calculated the sensitivity, specificity, positive and negative predictive values of a single RT-LAMP swab compared to RT-PCR, as per STARD guidelines. We also recorded vital signs of patients to correlate clinical and laboratory information.FindingsThe novel method accurately detected 8/10 PCR positive cases and identified a further 3 positive cases. Eight further cases were negative using both methods. Using repeated RT-PCR as a “gold standard”, the sensitivity and specificity of the novel test were 80% and 73% respectively. Positive predictive value (PPV) was 73% and negative predictive value (NPV) was 83%. We also observed hypothermia to be a significant early clinical sign in a number of COVID-19 patients in this setting.InterpretationRT-LAMP testing for SARS-CoV-2 was found to be promising, fast, easy to use and to work equivalently to RT-PCR methods. Definitive studies to evaluate this method in larger cohorts are underway. RT-LAMP has the potential to transform COVID-19 detection, bringing rapid and accurate testing to the point of care. This method could be deployed in mobile testing units in the community, care homes and hospitals to detect disease early and prevent spread.

Background Obstetrical brachial plexus palsy is a common birth injury to nerves passing through the brachial plexus that may result in structural and functional abnormalities. Individual joint trajectories from kinematic analyses have been used to evaluate the source and extent of abnormalities. Here, two summary measures of limb kinematics were utilized: 1) the Arm Profile Score summarizing upper limb joint kinematic abnormalities from a typical pattern across a task, and 2) the recently developed Multi-joint Coordination Measure using principal component analysis to characterize typical coordination of multiple joints throughout a task and compute deviations in time and space. Our aim was to compare these kinematic measures in persons with and without injury and relate these to clinical and functional scales. Methods 3D kinematic data from 10 upper limb joints were collected on 15 children and adolescents with obstetrical brachial plexus palsy and 21 controls during a reach-to-grasp task in both limbs. The two kinematic measures were computed and correlated with each other and the Mallet and ABILIHAND-Kids. Findings Both measures revealed that joint angles primarily contributing to shoulder and wrist motion were most prominently affected in the non-dominant limb in obstetrical brachial plexus palsy, with the Multi-joint Coordination Measure additionally indicating when in the motion coordination worsens. These were moderately interrelated but neither correlated with other scales. Interpretation The Multi-joint Coordination Measure, while related to the Arm Profile Score, may have additional utility for individualized treatment planning and evaluation of any motor task due to the unique spatial-temporal information provided.

Background. Locomotor training using treadmills or robotic devices is commonly utilized to improve gait in cerebral palsy (CP); however, effects are inconsistent and fail to exceed those of equally intense alternatives. Possible limitations of existing devices include fixed nonvariable rhythm and too much limb or body weight assistance. Objective: To quantify and compare effectiveness of a motor-assisted cycle and a novel alternative, an elliptical, in CP to improve interlimb reciprocal coordination through intensive speed-focused leg training. Methods: A total of 27 children with bilateral CP, 5 to 17 years old, were randomized to 12 weeks of 20 minutes, 5 days per week home-based training (elliptical = 14; cycle = 13) at a minimum of 40 revolutions per minute, with resistance added when speed target was achieved. Primary outcomes were self-selected and fastest voluntary cadence on the devices and gait speed. Secondary outcomes included knee muscle strength, and selective control and functional mobility measures. Results: Cadence on trained but not nontrained devices increased, demonstrating task specificity of training and increased exercise capability. Mean gait speed did not increase in either group, nor did parent-reported functional mobility. Knee extensor strength increased in both. An interaction between group and time was seen in selective control with scores slightly increasing for the elliptical and decreasing for the cycle, possibly related to tighter limb coupling with cycling. Conclusions: Task-specific effects were similarly positive across groups, but no transfer was seen to gait or function. Training dose was low (≤20 hours) compared with intensive upper-limb training recommendations and may be insufficient to produce appreciable clinical change.

Motor adaptation, or alteration of neural control in response to a perturbation, is a potential mechanism to facilitate motor learning for rehabilitation. Central nervous system deficits are known to affect locomotor adaptation; yet we demonstrated that similar to adults following stroke, children with unilateral brain injuries can adapt step length in response to unilateral leg weighting. Here, we extend our analysis to explore kinematic strategies underlying step length adaptation and utilize dynamical systems approaches to elucidate how neural control may differ in those with hemiplegic CP across legs and compared to typically developing controls. Ten participants with hemiplegic CP and ten age-matched controls participated in this study. Knee and hip joint kinematics were analyzed during unilateral weighting of each leg in treadmill walking to assess adaptation and presence and persistence of after-effects. Peak joint angle displacement was used to represent changes in joint angles during walking. We examined baseline and task-specific variability and local dynamic stability to evaluate neuromuscular control across groups and legs. In contrast to controls, children with unilateral CP had asymmetries in joint angle variability and local dynamic stability at baseline, showing increased variability and reduced stability in the dominant limb. Kinematic variability increased and local stability decreased during weighting of ipsilateral and contralateral limbs in both groups compared to baseline. After weight removal both measures returned to baseline. Analogous to the temporal-spatial results, children with unilateral CP demonstrated similar capability as controls to adapt kinematics to unilateral leg weighting, however, the group with CP differed across sides after weight removal with dominant limb after-effects fading more quickly than in controls. The change in kinematics did not completely return to baseline in the non-dominant limb of the CP group, producing a transient improvement in joint angle symmetry. Recent studies demonstrate that neural control of gait is multi-layered with distinct circuits for different types of walking and for each leg. Remarkably, our results demonstrate that children with unilateral brain injury retain these separate circuits for each leg during walking and, importantly, that those networks can be adapted independently from one another to improve symmetry in the short term.

BACKGROUND: Patients have high reinjury rates after ACL reconstruction. Small knee flexion angles and large peak posterior ground reaction forces in landing tasks increase ACL loading. QUESTIONS/PURPOSES: We determined the effects of a knee extension constraint brace on knee flexion angle, peak posterior ground reaction force, and movement speed in functional activities of patients after ACL reconstruction. PATIENTS AND METHODS: Six male and six female patients 3.5 to 6.5 months after ACL reconstruction participated in the study. Three-dimensional videographic and force plate data were collected while patients performed level walking, jogging, and stair descent wearing a knee extension constraint brace, wearing a nonconstraint brace, and not wearing a knee brace. Knee flexion angle at initial foot contact with the ground, peak posterior ground reaction force, and movement speed were compared across brace conditions and between genders. RESULTS: Wearing the knee extension constraint brace increased the knee flexion angle at initial foot contact for each activity when compared with the other two brace conditions. Wearing the knee extension constraint brace also decreased peak posterior ground reaction force during walking but not during jogging and stair descent. CONCLUSIONS: Although the knee extension constraint brace did not consistently reduce the peak posterior ground reaction force in all functional activities, it consistently increased knee flexion angle and should reduce ACL loading as suggested by previous studies. These results suggest the knee extension constraint brace has potential as a rehabilitation tool to alter lower extremity movement patterns of patients after ACL reconstruction to address high reinjury rates.

There are several advantages that functional near-infrared spectroscopy (fNIRS) presents in the study of the neural control of human movement. It is relatively flexible with respect to participant positioning and allows for some head movements during tasks. Additionally, it is inexpensive, light weight, and portable, with very few contraindications to its use. This presents a unique opportunity to study functional brain activity during motor tasks in individuals who are typically developing, as well as those with movement disorders, such as cerebral palsy. An additional consideration when studying movement disorders, however, is the quality of actual movements performed and the potential for additional, unintended movements. Therefore, concurrent monitoring of both blood flow changes in the brain and actual movements of the body during testing is required for appropriate interpretation of fNIRS results. Here, we show a protocol for the combination of fNIRS with muscle and kinematic monitoring during motor tasks. We explore gait, a unilateral multi-joint movement (cycling), and two unilateral single-joint movements (isolated ankle dorsiflexion, and isolated hand squeezing). The techniques presented can be useful in studying both typical and atypical motor control, and can be modified to investigate a broad range of tasks and scientific questions.

Treadmills provide a safe and efficient method for gait rehabilitation but treadmill based training paradigms have not been shown to create superior results when compared with traditional physical therapy methods such as overground training. One explanation for this may be that walking at a constant, fixed speed requires little mental engagement from the user, which has been postulated as a key factor in the success of motor learning. To increase mental engagement, we developed a user-driven treadmill control scheme. In this paper we use electroencephalography (EEG) to compare cortical activity during user-driven (active) walking with activity on a normal (passive) treadmill in nine healthy subjects. We used independent component analysis (ICA) to isolate brain activity from artifactual components. We fit equivalent dipole sources to each brain component and clustered these across subjects. Our analysis revealed that relative to the passive treadmill, active walking resulted in statistically significant decreases in spectral power, i.e. desynchronization, in the anterior cingulate, sensorimotor cortices, and posterior parietal lobe of the cortex. These results indicate that user-driven treadmills more fully engage the motor cortex and therefore could facilitate better training outcomes than a traditional treadmill.

A self-paced treadmill that can simulate overground walking has the potential to improve the effectiveness of treadmill training for gait rehabilitation. We have implemented a self-paced treadmill without the need for expensive equipment such as a motion capture system and an instrumented treadmill. For this, an inexpensive depth sensor, ASUS XtionTM, substitutes for the motion capture system, and a low-cost commercial treadmill is considered as the platform of the self-paced treadmill. The proposed self-paced treadmill is also convenient because the depth sensor does not require markers placed on user's body. Through pilot tests with two healthy subjects, it is quantitatively and qualitatively verified that the proposed self-paced treadmill achieves similar performance as one which utilizes a commercial motion capture system (VICON) as well as an instrumented treadmill.

Treadmill-based locomotor training should simulate overground walking as closely as possible for optimal skill transfer. The constant speed of a standard treadmill encourages automaticity rather than engagement and fails to simulate the variable speeds encountered during real-world walking. To address this limitation, this paper proposes a user-driven treadmill velocity control scheme that allows the user to experience natural fluctuations in walking velocity with minimal unwanted inertial force due to acceleration/deceleration of the treadmill belt. A smart estimation limiter in the scheme effectively attenuates the inertial force during velocity changes. The proposed scheme requires measurement of pelvic and swing foot motions, and is developed for a treadmill of typical belt length (1.5 m). The proposed scheme is quantitatively evaluated here with four healthy subjects by comparing it with the most advanced control scheme identified in the literature.

Protein kinases play a critical role in almost every cellular regulatory process and have been identified as key players in diseases such as cancer and immune syndromes (1). For this reason, there has been substantial interest in the development of assays for protein kinases for use as both diagnostics and drug discovery tools (2). Although several assays exist for kinases, the most commonly used involve monitoring transfer of the γ-phosphoryl group from [γ-32P]ATP to a peptide substrate (3). These assays are cumbersome to implement because the unreacted [γ-32P]ATP needs to be separated from the phosphorylated peptide by use of separation techniques such as gel electrophoresis. In drug discovery programs, fluorescence resonance energy transfer (FRET) kinase assays are becoming increasingly popular because they are homogeneous, making them relatively easy to automate (4). In typical FRET assays, the anti-phosphotyrosine antibody and the substrate peptide are labeled with “donor” and “acceptor” fluorophores. On phosphorylation of the peptide, the anti-phosphotyrosine antibody binds to the peptide, and the two fluorophores are brought in close proximity to each other. Excitation of the acceptor fluorophore leads to energy transfer to the donor molecule, which emits fluorescence. The principal disadvantage of FRET-based assays is that they are difficult to configure because the two fluorophores need to be within a closely defined distance of each other. The Dual Phage technology is a new ultrasensitive biological amplification system of broad applicability that uses bacteriophages as …

The extent to which therapeutic, exercise or robotic devices can maximize gait function is a major unresolved issue in neurorehabilitation. Several factors may influence gait outcomes such as similarity of the task to overground walking, degree of coordination within and across limbs, and cycle-to-cycle variability in each device. Our objective was to compare lower extremity kinematics, coordination and variability during four locomotor tasks: overground walking, treadmill walking, elliptical training and stationary cycling in 10 non-disabled adults (6 male; mean age 22.7±2.9 yrs, range 20-29). All first performed four overground walking trials at self-selected speed with mean temporal-spatial data used to pace the other conditions. Joint positions, excursions, and the Gait Deviation Index (GDI) were compared across conditions to evaluate kinematic similarity. Time-series data were correlated within and across limbs to evaluate intralimb and interlimb coordination, respectively. Variability in cadence was quantified to assess how constrained the locomotor rhythm was compared to overground walking. Treadmill walking most closely resembled overground with GDI values nearly overlapping, reinforcing its appropriateness for gait training. Cycling showed the largest GDI difference from overground, with elliptical closer but still a significant distance from all three. Cycling showed greater hip reciprocation Cycling and elliptical showed stronger intralimb synergism at the hip and knee than the other two. Based on kinematics, results suggest that elliptical training may have greater transfer to overground walking than cycling and cycling may be more useful for enhancing reciprocal coordination. Further evaluation of these devices in neurological gait disorders is needed.

The most common functional motor goal of lower extremity rehabilitation is to improve walking ability. For reasons of feasibility, safety or intensity, devices are frequently used to facilitate or augment gait training. The objective of this study was to compare the muscle activity patterns of the rectus femoris and semitendinosus muscles during four conditions: overground walking, treadmill walking, stationary cycling, and elliptical training. Ten healthy adults (six male, four female; mean age 22.7±2.9 years, range 20-29) participated and surface electromyographic data were recorded. Linear envelope curves were generated and time normalized from 0 to 100% cycle. The mean plus three standard deviations from a static trial was used as the threshold for muscle activity. Repeated measures analysis of variance procedures were used to detect differences between conditions. Elliptical training demonstrated greater rectus femoris activity and greater rectus femoris/semitendinosus coactivation than all other conditions. Consistent with previous work, treadmill walking demonstrated greater rectus femoris activity than overground walking. Minimal differences in semitendinosus activation were observed between conditions, limited to lower peak activity during cycling compared to treadmill walking. These results provide normative values for rectus femoris and semitendinosus activation for different locomotor training methods and may assist in selecting the most appropriate training device for specific patients. Clinicians and researchers should also consider the kinematic and kinetic differences between tasks, which cannot necessarily be inferred from muscle activation patterns.

Detection of bacterial growth is used in the diagnosis and treatment of infectious disease, blood screening, food safety, product quality assurance, and life science research. The measurement of intracellular ATP content has long been the standard for rapid bacterial growth and viability measurement (1)(2)(3). The current methods used to detect ATP include luminescence generated by the enzyme system firefly luciferase/luciferin. The chemistry involved in this process is simple and can be used with a wide range of luminescence equipment, from handheld devices to sophisticated, laboratory-based instruments for high-throughput applications(4). We have developed an alternative, quantitative assay method for ATP based on a nucleic acid testing (NAT) format. During the past decade NAT has become the method of choice for bacterial identification (5). NAT hardware, such as thermal cyclers, isothermal instruments, and real-time/kinetic product detection systems, are now commonplace laboratory equipment. The new method, LiMA (Ligase Mediated ATP Amplification Assay)(6), uses DNA ligase, an ATP-requiring enzyme(7), to join 2 oligonucleotides in a nicked-DNA substrate and create a template that can be amplified in a DNA amplification reaction (Fig. 1⇓ ). Before the LiMA process, the ligase is treated with pyrophosphate to ensure that all the enzyme molecules are in the deadenylated form. Thus the ligase is inactive until it binds a molecule of ATP, which leads to the loss of the pyrophosphate moiety from ATP and the formation of a covalent enzyme—AMP intermediate linked to a lysine side-chain in the enzyme. In this reaction the enzyme becomes charged by an ATP molecule; essentially, a sample is scavenged for its ATP content. In the next step of the reaction the AMP nucleotide is transferred to the 5′ phosphate of the nicked-DNA strand, followed by attack on the AMP-DNA bond by the 3′-OH of …

There have been many reports describing the combination of immunoassay techniques and nucleic acid amplification using antibodies labeled directly with nucleic acids (the “direct” immuno-PCR approach) (1)(2)(3). The rationale behind immuno-PCR is to develop ultrahigh-sensitivity labels by exploiting the extremely high productivity of nucleic acid amplification techniques, coupled with highly sensitive approaches to detect the amplified material. Specific applications in which the use of ultrahigh-sensitivity immuno-PCR techniques have been proposed include prion protein detection, in which there is no nucleic acid associated with the infectious agent, and the detection of viral antigens in blood bank screening applications, in which there can be very little viral nucleic acid present at certain stages of the infection. Although the principle of immuno-PCR has been demonstrated in various research applications, there are continuing concerns over both the technical difficulty of linking nucleic acids directly to antibodies (or “adapter” proteins such as streptavidin) and the substantial backgrounds that can be generated by nonspecific binding of the nucleic acid–antibody conjugates to solid phases (2)(4). We have developed an alternative approach based on an “indirect” immuno-PCR technique that links ELISA techniques and PCR. Because this approach works in a completely different way to traditional immuno-PCR, it may circumvent some of the problems associated with the traditional approach. Here we present the proof of principle of our new approach. We used a double stranded 5′-phosphorylated DNA (dsDNA) substrate for alkaline phosphatase (AP). Shown in Fig. 1A⇓ is a schematic of the principle of the enzyme assay method. The procedure takes advantage of the substrate specificity of λ exonuclease for the 5′-phosphorylated form of dsDNA (5). In an immunometric assay, the AP in the antibody conjugate will remove the 5′ phosphate groups, and the DNA …

Background Balance problems are common in cerebral palsy (CP) but etiology is often uncertain. The classic Romberg test compares ability to maintain standing with eyes open versus closed. Marked instability without vision is a positive test and generally indicates proprioceptive loss. From previous work showing diminished hip joint proprioception in CP, we hypothesized that static and dynamic balance without vision (positive Romberg) would be compromised in CP. Methods Force plate sway and gait velocity data were collected using 3D motion capture on 52 participants, 19 with diplegic CP, 13 with hemiplegic CP, and 20 without disability. Center of mass (COM) and center or pressure (COP) velocity, excursion, and differences between COM and COP in AP and ML directions were computed from static standing trials with eyes open and closed. Mean gait velocity with and without dribble glasses was compared. Hip joint proprioception was quantified as the root mean square of magnitude of limb positioning errors during a hip rotation task with and without view of the limb. Mixed model repeated measures analysis of variance (ANOVA) was performed with condition as within-subject (EO, EC) and group as between-subject factors (hemiplegia, diplegia, controls). Sway characteristics and gait speed were correlated with proprioception values. Results Groups with CP had greater sway in standing with eyes open indicating that they had poorer balance than controls, with the deficit relatively greater in the ML compared to AP direction. Contrary to our hypothesis, the decrement with eyes closed did not differ from controls (negative Romberg); however, proprioception error was related to sway parameters particularly for the non-dominant leg. Gait speed was related to proprioception values such that those with worse proprioception tended to walk more slowly. Conclusions Postural instability is present even in those with mild CP and is yet another manifestation of their motor control disorder, the specific etiology of which may vary across individuals in this heterogeneous diagnostic category.

The misfolding of normal proteins and their subsequent aggregation in the brain is characteristic of a group of related neurologic diseases, the “protein conformational disorders”, including Parkinson disease, Alzheimer disease, and the transmissible spongiform encephalopathies (TSEs) (1). The development of highly sensitive and specific diagnostic assays for these diseases is a high priority as effective therapies become available or as food safety regulations mandate the removal of infected animals from the food chain. Current test development effort in the TSE field has been focused mainly on designing screening tests for the aggregated prion proteins in bovine, ovine, and cervid brain tissue. Several immunoassays for bovine spongiform encephalopathy (BSE) have been evaluated by the European Union (EU) (2), and three are now approved for routine use in testing laboratories. Those approved include two ELISAs in the microplate format and reagents for a Western blot analysis. The abnormal, or rogue, prion protein detected by these tests is considered to be the best indicator of TSE infection (2), but the anti-prion protein antibodies used in these assays cannot distinguish the normal, globular, proteinase K-sensitive (α-helix-rich) form of the prion protein (PrPsen) from the abnormal, aggregated, and relatively proteinase K-resistant form (PrPres), in which the secondary structure is dominated by β-sheet. Thus, a complex sample preparation procedure is required to eliminate the normal prion protein in the brain homogenate before immunoassay, usually involving a proteinase K digestion step …