Magnetomicrometry.

We live in an era of wearable sensing, where our movement through the world can be continuously monitored by devices. Yet, we lack a portable sensor that can continuously monitor muscle, tendon, and bone motion, allowing us to monitor performance, deliver targeted rehabilitation, and provide intuitive, reflexive control over prostheses and exoskeletons. Here, we introduce a sensing modality, magnetomicrometry, that uses the relative positions of implanted magnetic beads to enable wireless tracking of tissue length changes. We demonstrate real-time muscle length tracking in an in vivo turkey model via chronically implanted magnetic beads while investigating accuracy, biocompatibility, and long-term implant stability. We anticipate that this tool will lay the groundwork for volitional control over wearable robots via real-time tracking of muscle lengths and speeds. Further, to inform future biomimetic control strategies, magnetomicrometry may also be used in the in vivo tracking of biological tissues to elucidate biomechanical principles of animal and human movement.

Tonic-clonic seizure after unrecognized unintentional dural puncture.

Patients who suffer an unintentional dural puncture have a high risk of developing a post-dural puncture headache. Other neurologic complications have been reported, but seizure is rarely seen. We present a case of a 21-year-old primigravida who experienced an unrecognized unintentional dural puncture that ultimately resulted in a tonic-clonic seizure from intracranial hypotension one week following the dural breach. Her trachea was intubated and she was transferred to the intensive care unit. Two epidural blood patches, performed by neuroradiologists, were needed before the patient experienced complete resolution of her headache. During the re-admission, she also experienced a pulmonary embolus which further lengthened her hospital stay.

On prosthetic control: A regenerative agonist-antagonist myoneural interface.

Prosthetic limb control is fundamentally constrained by the current amputation procedure. Since the U.S. Civil War, the external prosthesis has benefited from a pronounced level of innovation, but amputation technique has not significantly changed. During a standard amputation, nerves are transected without the reintroduction of proper neural targets, causing painful neuromas and rendering efferent recordings infeasible. Furthermore, the physiological agonist-antagonist muscle relationships are severed, precluding the generation of musculotendinous proprioception, an afferent feedback modality critical for joint stability, trajectory planning, and fine motor control. We establish an agonist-antagonist myoneural interface (AMI), a unique surgical paradigm for amputation. Regenerated free muscle grafts innervated with transected nerves are linked in agonist-antagonist relationships, emulating the dynamic interactions found within an intact limb. Using biomechanical, electrophysiological, and histological evaluations, we demonstrate a viable architecture for bidirectional signaling with transected motor nerves. Upon neural activation, the agonist muscle contracts, generating electromyographic signal. This contraction in the agonist creates a stretch in the mechanically linked antagonist muscle, producing afferent feedback, which is transmitted through its motor nerve. Histological results demonstrate regeneration and the presence of the spindle fibers responsible for afferent signal generation. These results suggest that the AMI will not only produce robust signals for the efferent control of an external prosthesis but also provide an amputee's central nervous system with critical musculotendinous proprioception, offering the potential for an enhanced prosthetic controllability and sensation.

Issues in using whole slide imaging for diagnostic pathology: "routine" stains, immunohistochemistry and predictive markers.

The traditional microscope, together with the "routine" hematoxylin and eosin (H & E) stain, remains the "gold standard" for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of "stains" available. Because of the need for specific identification and even measurement of "biomarkers," immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.

Energy benchmarks for water clusters and ice structures from an embedded many-body expansion.

We show how an embedded many-body expansion (EMBE) can be used to calculate accurate ab initio energies of water clusters and ice structures using wavefunction-based methods. We use the EMBE described recently by Bygrave et al. [J. Chem. Phys. 137, 164102 (2012)], in which the terms in the expansion are obtained from calculations on monomers, dimers, etc., acted on by an approximate representation of the embedding field due to all other molecules in the system, this field being a sum of Coulomb and exchange-repulsion fields. Our strategy is to separate the total energy of the system into Hartree-Fock and correlation parts, using the EMBE only for the correlation energy, with the Hartree-Fock energy calculated using standard molecular quantum chemistry for clusters and plane-wave methods for crystals. Our tests on a range of different water clusters up to the 16-mer show that for the second-order Møller-Plesset (MP2) method the EMBE truncated at 2-body level reproduces to better than 0.1 mE(h)/monomer the correlation energy from standard methods. The use of EMBE for computing coupled-cluster energies of clusters is also discussed. For the ice structures Ih, II, and VIII, we find that MP2 energies near the complete basis-set limit reproduce very well the experimental values of the absolute and relative binding energies, but that the use of coupled-cluster methods for many-body correlation (non-additive dispersion) is essential for a full description. Possible future applications of the EMBE approach are suggested.

Isolation of bacterial strains able to metabolize lignin from screening of environmental samples.

AIMS: To develop a method to detect bacteria from environmental samples that are able to metabolize lignin. METHODS AND RESULTS: A previously developed UV-vis assay method for lignin degradation activity has been developed for use as a spray assay on agar plates. Nine mesophilic strains were isolated using this method from woodland soil incubated in enrichment cultures containing wheat straw lignocellulose: four Microbacterium isolates, two Micrococcus isolates, Rhodococcus erythropolis (all Actinobacteria) and two Ochrobactrum isolates (Alphaproteobacteria). Three thermotolerant isolates were isolated from the same screening method applied at 45°C to samples of composted wheat straw from solid-state fermentation: Thermobifida fusca and two isolates related to uncharacterized species of Rhizobiales and Sphingobacterium (Bacteroidetes), the latter strain showing tenfold higher lignin degradation activity than other isolates. The isolated strains were able to depolymerize samples of size-fractionated high molecular weight and low molecular weight Kraft lignin, and produced low molecular weight metabolites oxalic acid and protocatechuic acid from incubations containing wheat straw lignocellulose. CONCLUSIONS: A new method for the isolation of bacteria able to metabolize lignin has been developed, which has been used to identify 12 bacterial isolates from environmental sources. The majority of isolates cluster into the Actinobacteria and the Alphaproteobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Lignin-degrading bacterial strains could be used to convert lignin-containing feedstocks into renewable chemicals and to identify new bacterial lignin-degrading enzymes.

GSTPi-positive tumour microenvironment-associated fibroblasts are significantly associated with GSTPi-negative cancer cells in paired cases of primary invasive breast cancer and axillary lymph node metastases.

BACKGROUND: Glutathione S-transferase Pi (GSTPi) expression is one of the factors, which is known to be associated with development of resistance to chemotherapeutics in cancer patients, including those with breast cancer. Yet, its expression has been reported to be undetectable in cancer cells in high percent of patients with primary breast cancer. However, GSTPi expression in stromal cells in breast tumour microenvironment, namely cancer-associated fibroblast (CAF), which is recognised to have major roles in cancer progression, remains poorly reported. METHODS: The aim of the study was to determine the expression of GSTPi; vimetin, a fibroblast-associated cytoskeleton protein; and α-smooth muscle actin (α-SMA), a known marker of CAF in breast cancer tissue, by immunohistochemical staining method in consecutive histologic sections of formalin-fixed and paraffin-embedded tissue biopsy specimens from a cohort of 39 paired cases of patients with invasive breast cancer and the corresponding axillary lymph nodes metastases. RESULTS: Ductal and acinar luminal epithelial cells, myoepithelial cells and surrounding fibroblasts exhibited a homogeneous cytoplasmic reactivity with anti-GSTPi antibody in 11 of 11 cases of benign breast tissue biopsies. The vimentin-positive fibroblasts were unreactive with anti-α-SMA antibody. Loss of GSTPi expression was observed in breast cancer cells, at both the primary and metastatic sites, in 31 of 39 paired cases, as compared with benign breast epithelial cells (Fisher's exact test P<0.001). A significant association was observed between GSTPi-positive, vimentin-positive and α-SMA-positive fibroblast in tumour microenvironment at both sites. CONCLUSION: This is an original report of demonstration of a significance association between tumour microenvironment-associated GSTPi-positive CAF (vimentin/α-SMA-positive) and the GSTPi-negative cancer cells in paired cases of primary invasive breast cancer and the corresponding axillary lymph nodes metastases.

Tissue engineered tumor models.

Many research programs use well-characterized tumor cell lines as tumor models for in vitro studies. Because tumor cells grown as three-dimensional (3-D) structures have been shown to behave more like tumors in vivo than do cells growing in monolayer culture, a growing number of investigators now use tumor cell spheroids as models. Single cell type spheroids, however, do not model the stromal-epithelial interactions that have an important role in controlling tumor growth and development in vivo. We describe here a method for generating, reproducibly, more realistic 3-D tumor models that contain both stromal and malignant epithelial cells with an architecture that closely resembles that of tumor microlesions in vivo. Because they are so tissue-like we refer to them as tumor histoids. They can be generated reproducibly in substantial quantities. The bioreactor developed to generate histoid constructs is described and illustrated. It accommodates disposable culture chambers that have filled volumes of either 10 or 64 ml, each culture yielding on the order of 100 or 600 histoid particles, respectively. Each particle is a few tenths of a millimeter in diameter. Examples of histological sections of tumor histoids representing cancers of breast, prostate, colon, pancreas and urinary bladder are presented. Potential applications of tumor histoids include, but are not limited to, use as surrogate tumors for pre-screening anti-solid tumor pharmaceutical agents, as reference specimens for immunostaining in the surgical pathology laboratory and use in studies of invasive properties of cells or other aspects of tumor development and progression. Histoids containing nonmalignant cells also may have potential as "seeds" in tissue engineering. For drug testing, histoids probably will have to meet certain criteria of size and tumor cell content. Using a COPAS Plus flow cytometer, histoids containing fluorescent tumor cells were analyzed successfully and sorted using such criteria.

Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II.

Immunohistochemistry is entering its fourth decade of use on formalin-fixed paraffin-embedded tissues. Over this period the method has evolved to become a major part of the practice of diagnostic surgical pathology worldwide. From the beginning immunohistochemistry has been adapted to provide a range of markers of cell lineage and tissue type, with particular application to the diagnosis and classification of tumours. In this modality immunohistochemical methods were employed simply as 'special stains', the results of which were evaluated qualitatively by the pathologist, as for any other stain. More recently, attention has shifted to the demonstration of prognostic markers in tumour cells, driven by the advent of molecular biology and the discovery of numerous regulatory molecules, coupled with manufacture of the corresponding specific antibodies. Immunohistochemistry has rapidly adapted to this new use, but in so doing the demand for quantification has become paramount; it is no longer enough that the 'stain' is there; rather it is a question of 'How much is there?'. This review explores the limitations of immunohistochemistry when employed in a semiquantitative mode, and explores the possibility of fulfilling the full potential of immunohistochemistry, as a true quantitative immunoassay applied in a tissue section environment.

Standardization in immunohistochemistry: the role of antigen retrieval in molecular morphology.

Molecular morphology seeks to integrate the traditional morphologic criteria of surgical pathology with immunohistochemical and in situ hybridization techniques that allow demonstration of a variety of molecules, proteins, RNA and DNA in a tissue section. While immunohistochemistry has proven to be successful for demonstrating lineage related biomarkers of value for diagnosis and classification of tumors, concerns have been raised periodically about validation of reagents, overall reproducibility of the staining method, and interpretation of results. These concerns have been heightened by the burgeoning interest in prognostic markers, where the question extends beyond a relatively simple positive or negative result to an absolute need for quantification of the staining result; not only is it positive, but how much is there? In this presentation at the Annual Meeting of the Biological Stain Commission in June, 2005, I advocate a total test approach that requires systematic attention to pre-analytic, analytic, and post-analytic issues. The approach encompasses all aspects of test performance from specimen acquisition, through fixation, antigen retrieval, processing, staining, interpretation, and reporting of results. A similar systematic approach also may be adopted for in situ hybridization methods, which have performance requirements that in many ways parallel immunohistochemistry.

Assessment of the elution of charcoal, cellulose acetate, and other particles from cigarettes with charcoal and activated charcoal/resin filters.

This experiment was designed to study the release of cellulose acetate fibers, charcoal, and other particles from cigarettes with charcoal and activated charcoal/resin filters. For the first time in such studies, efforts were made to identify the particles that were eluted using other analytical techniques in addition to light microscopy. Other corrective measures were also implemented. During the studies it was found that trimming of larger filters to fit smaller filter housings introduced cellulose acetate-like particles from the fibers of the filter material. Special, custom made-to-fit filters were used instead. Tools such as forceps that were used to retrieve filters from their housings were also found to introduce fragments onto the filters. It is believed that introduction of such debris may have accounted for the very large number of cellulose acetate and charcoal particles that had been reported in the literature. Use of computerized particle-counting microscopes appeared to result in excessive number of particles. This could be because the filter or smoke pads used for such work do not have the flat and level surfaces ideal for computerized particle-counting microscopes. At the high magnifications that the pads were viewed for particles, constant focusing of the microscope would be essential. It was also found that determination of total particles by using extrapolation of particle count by grid population usually gave extremely high particle counts compared to the actual number of particles present. This could be because particle distributions during smoking are not uniform. Lastly, a less complex estimation of the thickness of the particles was adopted. This and the use of a simple mathematical conversion coupled with the Cox equation were utilized to assess the aerodynamic diameters of the particles. Our findings showed that compared to numbers quoted in the literature, only a small amount of charcoal, cellulose acetate shards, and other particles are released. It was also shown that those particles would have a low likelihood of reaching the lung.

Different localization of dendritic cell reservoirs in human immunodeficiency virus-1 subtype B versus subtype E-infected lymph nodes.

The presence of p24 protein was studied in lymph nodes from human immunodeficiency virus (HIV)-positive patients affected by persistent generalized lymphadenopathy. Paraffin-embedded lymph node sections from 50 HIV-1 subtype E-infected lymph nodes from patients in Thailand and 25 HIV-1 presumably subtype B-infected lymph nodes from patients in the United States were immunostained with p24 HIV major core and capsid monoclonal antibodies using the streptavidin-biotin immunoperoxidase technique. Positivity for HIV p24 protein was detected in 20 of 22 HIV-1 subtype B infected nodes in which lymphoid follicles were present, with p24 staining demonstrating a reticular pattern within the germinal centers. Interestingly, no case from 50 clade E-infected lymph nodes containing lymphoid follicles had such a reticular pattern in the germinal centers. This difference could be explained by differential infection of subsets of dendritic cells by the two HIV-1 clades, or perhaps by different routes of initial HIV-1 transmission.

Molecular morphology of Hodgkin lymphoma.

No other disease entity has provided greater impetus for the development of the concept of "molecular morphology" than Hodgkin lymphoma. Efforts to understand the etiology of this enigmatic disease have stimulated the application and refinement of almost every mode of biomedical scientific exploration. Notwithstanding the vast amount of data generated, much is still unknown about this remarkable disease, serving as an ongoing inducement to the development and application of new technologies for the analysis of cells and molecules in a morphologic environment.

Antigen retrieval techniques: current perspectives.

Development of the antigen retrieval (AR) technique, a simple method of boiling archival paraffin-embedded tissue sections in water to enhance the signal of immunohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide application of IHC in research and clinical pathology. On the basis of thousands of articles and many reviews, a book has recently been published that summarizes basic principles for practice and further development of the AR technique. Major topics with respect to several critical issues, such as the definition, application, technical principles, and further studies of the AR technique, are highlighted in this article. In particular, a further application of the heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to proteins, and standardization of routine IHC based on the AR technique in terms of a test battery approach, are also addressed. Furthermore, understanding the mechanism of the AR technique may shed light on facilitating the development of molecular morphology.

Antigen retrieval immunohistochemistry and molecular morphology in the year 2001.

The impact of the antigen retrieval (AR) technique upon diagnostic immunohistochemistry (IHC) and upon research has been demonstrated by numerous of articles and more than a dozen major reviews. The specific aim of this survey of the field is to examine potential new approaches to retrieval of nucleic acid and protein from archival paraffin-embedded tissue for molecular biology-based diagnostic procedures that form the basis of the emerging field of molecular morphology. Any new approach must incorporate the principles of standardization and improved reproducibility. The ultimate goal will be to understand the mechanisms of fixation (by formalin) and "unfixation" (by AR). In the interim, the diligent application of practical procedures that have been shown to be tried and true is the least that we must demand from ourselves and our laboratories.

Evaluation of JPEG and wavelet compression of body CT images for direct digital teleradiologic transmission.

PURPOSE: To determine acceptable levels of JPEG (Joint Photographic Experts Group) and wavelet compression for teleradiologic transmission of body computed tomographic (CT) images. MATERIALS AND METHODS: A digital test pattern (Society of Motion Picture and Television Engineers, 512 x 512 matrix) was transmitted after JPEG or wavelet compression by using point-to-point and Web-based teleradiology, respectively. Lossless, 10:1 lossy, and 20:1 lossy ratios were tested. Images were evaluated for high- and low-contrast resolution, sensitivity to small signal differences, and misregistration artifacts. Three independent observers who were blinded to the compression scheme evaluated these image quality measures in 20 clinical cases with similar levels of compression. RESULTS: High-contrast resolution was not diminished with any tested level of JPEG or wavelet compression. With JPEG compression, low-contrast resolution was not lost with 10:1 lossy compression but was lost at 3% modulation with 20:1 lossy compression. With wavelet compression, there was loss of 1% modulation with 10:1 lossy compression and loss of 5% modulation with 20:1 lossy compression. Sensitivity to small signal differences (5% and 95% of the maximal signal) diminished only with 20:1 lossy wavelet compression. With 10:1 lossy compression, misregistration artifacts were mild and were equivalent with JPEG and wavelet compression. Qualitative clinical findings supported these findings. CONCLUSION: Lossy 10:1 compression is suitable for on-call electronic transmission of body CT images as long as original images are subsequently reviewed.

Kimura's disease with oral ulcers: response to pentoxifylline.

We describe a man with Kimura's disease whose presentation included lymphadenopathy and cutaneous nodules, but was most distinctive for painful oral ulcerations. His lesions showed an initially moderate, but ultimately minimal response to monthly triamcinolone injections. With oral pentoxyifylline, he showed resolution of all of his lesions for 14 months. On cessation of his treatment, his disease flared for 3 months. When pentoxyifylline was restarted, his lesions regressed again within 4 weeks. We review the literature on Kimura's disease.