Tongue reconstruction: Rebuilding mobile three-dimensional structures from immobile two-dimensional substrates, a fresh cadaver study.

OBJECTIVE: To determine the two-dimensional (2D) characteristics of flaps necessary to create three-dimensional (3D) tongue anatomy. METHODS: Dissection of 11 fresh, nonpreserved human cadavers was performed. Six defects in each were created: total tongue, total oral tongue, hemiglossectomy, oral hemiglossectomy, total base of tongue, and hemi-base of tongue. The resections were debulked to create flat, 2D mucosal flaps. The dimensions and shapes of these flaps were determined. RESULTS: Each specimen showed consistent dimensions and geometry between cadavers. The total tongue was pear-shaped, the total oral tongue was egg-shaped, the oral hemi-tongue was bullet-shaped, the hemi-tongue resembled a dagger, the total base of tongue was rectangular, and the hemi-base of tongue was hour-glass shaped. CONCLUSION: Typical dimensions and shapes of common tongue defects were determined. It is conceivable that customizing reconstructive flaps based on these data will increase the accuracy of neo-tongue reconstruction, and thus, improve functional outcomes.

Transoral robotic surgery for oropharyngeal cancer: patient selection and special considerations.

The increasing incidence of oropharyngeal squamous cell carcinoma (OPSCC) emphasizes the importance of optimizing treatment for the disease. Historical protocol has utilized definitive radiation and invasive open procedures; these techniques expose the patient to significant risks and morbidity. Transoral robotic surgery (TORS) has emerged as a therapeutic modality with promise. Here, the literature regarding proper patient selection and other considerations for this procedure was reviewed. Multiple patient and tumor-related factors were found to be relevant for successful use of this treatment strategy. Outcomes regarding early and advanced-stage OPSCC were analyzed. Finally, the literature regarding use of TORS in three distinct patient populations, individuals with primary OPSCC, carcinoma of unknown primary and those with recurrent OPSCC, was examined.

Predictors of returns to the emergency department after head and neck surgery.

BACKGROUND: Thirty-day hospital readmissions have become a measure of quality of care. Many readmissions enter through the emergency department. The purposes of this study were to determine the rate, risk factors, and costs of 30-day returns to the emergency department (30dEDRs) after head and neck surgery. METHODS: All adult patients undergoing head and neck surgery at the University of Florida from 2012 to 2014 were reviewed. Univariate and multivariate logistic regression analyses were performed to identify risk factors for 30dEDRs. RESULTS: We found 1065 patients who underwent 1173 procedures. There were 88 cases (7.5%) that resulted in 30dEDRs and 55 patients (4.7%) who had 30-day unplanned readmissions (30dURs). Significant predictors of 30dEDRs included: smoking; hypothyroidism; and intensive care unit (ICU) stays. Significant predictors of readmission from an emergency department visit were Charlson Comorbidity Index (CCI) and cancer stage. Total costs of 30dEDRs and any subsequent readmissions topped $500 000. CONCLUSION: The rate of 30dEDRs after head and neck surgery is low; however, these visits increase the hospitals' financial burden as well as patient morbidity. Predictors of 30dEDRs may be utilized to formulate preventative measures.

The recent medicinal chemistry development of Jak2 tyrosine kinase small molecule inhibitors.

Since the discovery of the Jak2-V617F mutation as the causative agent in a large number of myeloproliferative neoplasms (MPNs), there has been a drive to develop Jak2 specific inhibitors that can be used in therapy for MPN patients and other Jak2-related pathologies. Over the past few years, a number of research groups have sought to develop Jak2 tyrosine kinase inhibitors. These compounds are currently in pre-clinical or clinical trials. Unfortunately, there is still a need for more potent, specific, and orally bioavailable drugs to treat these diseases. Within the past twelve months, a variety of medicinal chemistry techniques have produced several lead compounds that exhibit promising Jak2 inhibitory properties. The majority of these inhibitors target the Jak2 kinase domain in general and the ATP-binding pocket in particular. In this review, we summarize these studies and discuss the structure activity relationship (SAR) properties of several compounds. As we learn more about the key structural components that provide potency and specificity in Jak2 inhibition, we will come closer to finding suitable treatment options for individuals suffering from Jak2-mediated pathologies.

Orphan kinesin NOD lacks motile properties but does possess a microtubule-stimulated ATPase activity.

NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NOD(DTW) and NOD("DR2")) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport.

Structural changes in the neck linker of kinesin explain the load dependence of the motor's mechanical cycle.

The two-headed motor protein kinesin hydrolyzes ATP and moves on microtubule tracks towards the plus end. The motor develops speeds and forces of the order of hundreds of nanometers per second and piconewtons, respectively. Recently, the dependence of the velocity, the dissociation rate and the displacement variance on the load and the ATP concentration were measured in vitro for individual kinesin molecules (Coppin et al., 1997; Visscher et al., 1999) over a wide range of forces. The structural changes in the kinesin motor that drive motility were discovered by Rice et al. (1999). Here we present a phenomenological model for force generation in kinesin based on the bi-stable, nucleotide-dependent behavior of the neck linker. We demonstrate that the model explains the mechanical, kinetic and statistical (experimental) data of Coppin et al. (1997). We also discuss the relationship between the model results and experimental data of Visscher et al. (1999).

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules.

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

Microinstrument gradient-force optical trap.

A micromachined fiber-optic trap is presented. The trap consists of four single-mode, 1064-nm optical intersection. The beam fibers mounted in a micromachined silicon and glass housing. Micromachining provides the necessary precision to align the four optical fibers so that the outputs have a common intersection forms a strong three-dimensional gradient-force trap with trapping forces comparable with that of optical tweezers. Characterization of the multibeam fiber trap is illustrated for capture of polystyrene microspheres, computer simulations of the trap stiffness, and experimental determination of the trapping forces.

Kinesin force generation measured using a centrifuge microscope sperm-gliding motility assay.

To measure force generation and characterize the relationship between force and velocity in kinesin-driven motility we have developed a centrifuge microscope sperm-gliding motility assay. The average (extrapolated) value of maximum isometric force at low kinesin density was 0.90 +/- 0.14 pN. Furthermore, in the experiments at low kinesin density, sperm pulled off before stall at forces between 0.40 and 0.75 pN. To further characterize our kinesin-demembranated sperm assay we estimated maximum isometric force using a laser trap-based assay. At low kinesin density, 4.34 +/- 1.5 pN was the maximum force. Using values of axoneme stiffness available from other studies, we concluded that, in our centrifuge microscope-based assay, a sperm axoneme functions as a lever arm, magnifying the centrifugal force and leading to pull-off before stall. In addition, drag of the distal portion of the axoneme is increased by the centrifugal force (because the axoneme is rotated into closer proximity to the glass surface) and represents an additional force that the kinesin motor must overcome.

A bipolar kinesin.

Chromosome segregation during mitosis depends on the action of the mitotic spindle, a self-organizing, bipolar protein machine which uses microtubules (MTs) and their associated motors. Members of the BimC subfamily of kinesin-related MT-motor proteins are believed to be essential for the formation and functioning of a normal bipolar spindle. Here we report that KRP130, a homotetrameric BimC-related kinesin purified from Drosophila melanogaster embryos, has an unusual ultrastructure. It consists of four kinesin-related polypeptides assembled into a bipolar aggregate with motor domains at opposite ends, analogous to a miniature myosin filament. Such a bipolar 'minifilament' could crosslink spindle MTs and slide them relative to one another. We do not know of any other MT motors that have a bipolar structure.

Rigorous analysis of light diffraction ellipsometry by striated muscle fibers.

A rigorous analysis of both the transverse electric and the transverse magnetic modes of light diffracted from a muscle fiber is performed. From the expressions of electromagnetic field components, ellipsometry parameters, differential field ratio, r, and birefringence, delta n, have been obtained. A theoretical formulation that introduces myofibril skew planes and a randomization factor about the average skew plane yields a relationship that shows good fit to experimental data of Chen et al. (Biophys. J. 56:595, 1989) and Burton et al. (J. Muscle Res. Cell Motil. 11:258, 1990). Using indices of refraction within each of the regions of the sarcomeric unit that are consistent with our knowledge of the molecular structure of the sarcomere in the analysis, it is shown that the transition from the rigor state to the resting state leads to as much as a approximately 13% decrease in the r-value and an equally significant change in delta n.

Force-velocity relationships in kinesin-driven motility.

Kinesin is a microtubule-based motor protein that uses energy released from Mg-ATP hydrolysis to generate force for the movement of intracellular membranes towards the fast-growing (plus) ends of microtubule tracks in cells. Kinesin-driven microtubule movement can be visualized and quantified using light microscope motility assays but our understanding of how kinesin generates force and motion is incomplete. Here we report the use of a centrifuge microscope to obtain force-velocity curves for kinesin-driven motility and to estimate that the maximal isometric force generated per kinesin is 0.12 +/- 0.03 pN per molecule.

Rigorous analysis of light diffraction by a striated muscle fibre.

A rigorous theory describing the diffraction of light by a muscle fibre has been formulated. The basis of this analysis is the rigorous coupled-wave approach of T. K. Gaylord & M. G. Moharam (Proc. IEEE 73, 894 (1985)); however, we obtain here a closed-form analytical result that is both mathematically simple and physically easy to understand. We have compared our results on striated muscle fibres with the analytical results obtained by A. F. Huxley (Proc. R. Soc. Lond. B 241, 65 (1990)) using the normal mode approach, and with those obtained by R. A. Thornhill, N. Thomas & N. Berovic (Eur. Biophys. J. 20, 87 (1991)) using a multiwave first-order coupled-wave approximation. For an equivalent set of assigned fibre parameters, our results are consistent with these mentioned. Extension of this analysis to a fibre with different structures showed that the differences in diffraction efficiencies of different orders for a frog skeletal fibre and for an insect flight fibre are clear; the sensitivity to distinct structural organization of the fibre is very good.

Isolation of a sea urchin egg kinesin-related protein using peptide antibodies.

To understand the roles of kinesin and its relatives in cell division, it is necessary to identify and characterize multiple members of the kinesin superfamily from mitotic cells. To this end we have raised antisera to peptides corresponding to highly conserved regions of the motor domains of several known members of the kinesin superfamily. These peptide antibodies react specifically with the motor domains of kinesin and ncd protein, as expected, and they also react with several polypeptides (including kinesin heavy chain) that cosediment with microtubules (MTs) precipitated from AMPPNP-treated sea urchin egg cytosol. Subsequent fractionation of ATP eluates of these MTs yields a protein of relative molecular mass 330 x 10(3) that behaves as a complex of three polypeptides that are distinct from conventional kinesin subunits or fragments thereof. This complex contains 85 kDa and 95 kDa polypeptides, which react with our peptide antibodies, and a 115 kDa polypeptide, which does not. This triplet of polypeptides, which we refer to as KRP(85/95), binds to purified sea urchin egg tubulin in an AMPPNP-enhanced, ATP-sensitive manner and induces the formation of microtubule bundles. We therefore propose that the triplet corresponds to a novel sea urchin egg kinesin-related protein.

The molecular origin of birefringence in skeletal muscle. Contribution of myosin subfragment S-1.

The state of optical polarization of He-Ne laser light diffracted by single skinned frog skeletal muscle fibers has been determined after decoration of the thin filaments of rigor fibers with exogenous S-1. Light on the first diffraction order was analyzed using optical ellipsometry for changes occurring in total birefringence (delta nT) and total differential field ratio (rT) and the experimental results compared with theoretical predictions. Fibers were examined with SDS-gel electrophoresis and electron microscopy as independent assays of S-1 binding. The binding of S-1 to the thin filaments caused a significant increase in rT and a small but significant decrease in delta nT. Release of bound exogenous S-1 with magnesium pyrophosphate demonstrated that the effect of S-1 on the optical parameters was reversible and both electrophoresis and electron microscopy demonstrated the presence of S-1 specifically bound to the thin filaments. Model simulations based on the theory of Yeh, Y., and R. Baskin (1988. Biophys. J. 54:205-218) showed that the values of delta nT and rT were sensitive to the axial bonding angle of exogenous S-1 as well as to the volume fraction of added S-1. Analysis of the data in light of the model showed that an average axial S-1 binding angle of 68 degrees +/- 7 degrees best fit the data.

Diffraction ellipsometry studies of osmotically compressed muscle fibers.

Microstructural features of relaxed, skinned muscle fibers compressed with polyvinylpyrrolidone were examined by optical diffraction ellipsometry. This technique is sensitive to the optical anisotropy within the muscle, including that due to intrinsic properties of the protein molecules as well as that due to the regular arrangement of proteins in the surrounding medium. The change in polarization state of light after interacting with the muscle is described by the differential field ratio (DFR) and birefringence (delta n). Compression of single fibers (sarcomere length = 2.6 microns) with 0%-21% polyvinylpyrrolidone caused an increase of up to 23% and 31% for DFR and delta n, respectively. The largest increase in both parameters occurred at intermediate sarcomere lengths. Theoretical modelling of the results suggest that the average S-1 tilt angle may be reduced upon compression of the filament lattice. This is supported by experiments in which S-1 was enzymatically cleaved with alpha-chymotrypsin. Separate experiments comparing fibers with intact membranes and skinned fibers compressed to an equivalent lattice spacing showed little difference in DFR or delta n.

Invasive pulmonary penicilliosis: successful therapy with amphotericin B.

We have described a patient with invasive pulmonary penicilliosis, documented by thoracotomy and cured with amphotericin B. Penicillium sp isolates in immunosuppressed patients should not be disregarded without a thorough investigation, especially if normally sterile sites are involved. Amphotericin B therapy may be successful, especially if accompanied by a reversal or moderation of immunosuppression.