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.

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.

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.

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.

First example of familial posttransfusion purpura in two PlA1-negative sisters.

Posttransfusion purpura (PTP) (platelet count 5000/microliter) was diagnosed in a female patient (never transfused, gravida IV, para IV) 1 week after transfusion for hysterectomy in 1978. She did not respond to pooled random-donor platelets but recovered following a single plasma exchange. Her platelets were PlA1 negative, and her plasma contained potent anti-PlA1. In 1986, her sister (never transfused, gravida III, para III) developed PTP (platelet counts 5-15,000/microliter) following surgery-associated transfusion. She did not respond to pooled random-donor platelets. Platelet-associated IgG was markedly elevated (5365) molecules/platelet; normal, less than 660); her plasma contained a potent platelet antibody with anti-PlA1 specificity. Her platelets were subsequently shown to be PlA1 negative. The platelet count did not rise above 30,000 per microliter, despite 3 days of high-dose methylprednisolone sodium succinate and 2 weeks of prednisone (80 mg/day). Later, her platelet count increased and remained normal after steroids were discontinued. The two sisters proved to be HLA-identical, and each possessed one haplotype carrying the DR3 marker, which has been implicated as a risk factor in neonatal alloimmune thrombocytopenia associated with anti-PlA1.

Optical depolarization changes in single, skinned muscle fibers. Evidence for cross-bridge involvement.

Optical ellipsometry studies of single, skinned muscle fibers conducted on the diffraction orders have yielded spectra that are sensitive to the state of the fiber. The linearly polarized light field vector becomes elliptically polarized as it passes through the fiber and may be collected at the diffraction orders. Fibers that have been subjected to extraction of myosin (0.6 M KCl) retain a weak diffraction pattern and exhibit a substantially decreased depolarization of incident linearly polarized light. A significant decrease in polarization is seen in skinned fibers that are subject to an increase in pH from 7.0 to 8.0. This increase in pH results in a decrease of approximately 30% in the depolarization angle of single fibers. The major decrease in depolarization angle that we observe at pH 8.0 is consistent with the notion that as cross-bridges move out from the shaft of the thick filament, their ability to cause depolarization of the incident linearly polarized light decreases. This interpretation is also consistent with the work of Ueno and Harrington where the decrease in the ability to cross-link S-1 and S-2 to the thick filament at pH 8.2 suggests cross-bridge movement away from the thick filament. A large decrease in birefringence, seen after treatment of skinned fibers with alpha-chymotrypsin, appears to be related to the breakdown of myosin into rod, S-1, heavy meromyosin, and light meromyosin.

Light diffraction patterns and sarcomere length variation in striated muscle fibers of Limulus.

Light diffraction patterns produced by Limulus striated muscle fibers were examined. Segments of fibers were glycerinated, fixed or bathed in relaxing solution. Profiles of the intensity of a diffracted order vs. the angle of incidence of the laser beam often exhibited narrow peaks with the fiber at rest length. The incident angles at which the intensity of left and right orders is greatest are used to calculate the sarcomere length, supporting the notion that regions of the fiber are organised into Bragg reflecting planes. These profiles developed subpeaks and broadened upon stretch of the fiber. The broad angle scan profiles are suggested to result from a decrease in the regular packing of myofibrils as the fiber is lengthened. The angular width of the subpeaks is used to estimate the thickness of clusters of myofibrils. The variation in sarcomere length along the fiber, as determined by the 0th to 1st diffraction order spacing, was dependent upon the fiber preparation. Glycerinated fibers and those bathed in relaxing solution showed more variation than fixed fibers. The variation of sarcomere length is compared to the variation in thick filament lengths in Limulus reported by Dewey et al. (1982). These results are compared to those obtained from frog fiber segments.

Isolation and characterization of sarcoplasmic reticulum from normal and dystrophic chicken.

Purified sarcoplasmic reticulum vesicles from normal and dystrophic chicken skeletal muscle have been isolated by a procedure employing pressure disruption of a microsomal suspension. The dystrophic sarcoplasmic reticulum (SR) exhibited reduced Ca++ transport and phosphoenzyme formation, but the Ca++-ATPase activity was normal. Normal and dystrophic SR showed similar lipid profiles, except for a significant increase in free fatty acids in the dystrophic SR. Investigations involving the interaction of oleic acid with normal SR showed fatty acids can induce conditions similar to those found in dystrophic SR.

Depolarization spectrum of diffracted light from muscle fiber. The intrinsic anisotropy component.

The depolarization signal of the diffraction patterns from muscle fibers includes information that differs from that of transmission birefringence experiments. Although both the birefringence studies and the phase shift studies of Yeh et al. (Yeh, Y, and G. Pinsky, 1983, Biophys. J., 42:83-90; Yeh, Y., M. E. Corcoran, R. J. Baskin, and R. L. Lieber, 1983, Biophys. J., 44:343-351) include inseparable intrinsic and form contributions, the present analysis shows that the magnitude of the E-field components of diffracted light is affected only by the intrinsic contribution. We have analyzed the amplitude portion of the data of which the phase shift portion had previously been reported (Yeh, Y., M. E. Corcoran, R. J. Baskin, and R. L. Lieber, 1983, Biophys. J., 44:343-351). For the relaxed-to-rigor transition, these field amplitudes also exhibit changes when ATP concentration is decreased. The observed decrease in optical depolarization upon rigor is consistent with the idea that optically anisotropic elements move away from the myosin thick filament under such conditions.

Stereological analysis of transverse tubules and sarcoplasmic reticulum isolated from normal and dystrophic skeletal muscle.

Vesicles isolated from the transverse tubules and sarcoplasmic reticulum of normal and dystrophic chicken skeletal muscle were analyzed for enzymatic activity and examined following freeze-fracture. A stereological procedure was used to determine particle density distributions on the resulting membrane fracture faces. The particle densities measured in this investigation were compared with those of an earlier study on intact muscle. Isolated sarcoplasmic reticulum vesicles showed a characteristically high P-face (cytoplasmic leaflet) particle density (5108 +/- 169 particles/micron2) and a low E-face (luminal leaflet) particle density (505 +/- 57 particles/micron2). Transverse tubule fractions showed a high E-face particle density (2346 +/- 179 particles/mu2) as well as a substantial P-face particle density (1019 +/- 129 particles/micron2). The high transverse tubule E-face particle density represents a characteristic morphological feature in the same way that the very high P-face particle density is characteristic of sarcoplasmic reticulum membranes. The major morphological alteration in dystrophic membranes was a shift in the E-face particle density distribution of isolated transverse tubules to a lower average particle density. (The E-face particle density of sarcoplasmic reticulum fractions showed no differences.)

Optical depolarization changes on the diffraction pattern in the transition of skinned muscle fibers from relaxed to rigor state.

Light diffraction spectra from single or small bundles of skinned striated muscle fibers show large changes in polarization properties when muscles are placed into rigor. The technique of combining optical diffraction and ellipsometry measurements has previously been shown by Yeh and Pinsky to be a sensitive probe of periodic anisotropic regions of the fiber. In the present work, using this method, the observed spectrum shows marked decrease in the measured phase angle, delta, as the fiber approaches the rigor state. The degree of phase angle change is a function of sarcomere length: Maximum overlap of approximately 2.3 microns gives the most change in delta a delta delta R-R approximately 35 degrees decrease for a bundle of three fibers. At a sarcomere length of 2.9 microns this delta delta R-R value is only 10 degrees. At a nonoverlapping length of approximately 3.8 microns, delta does not vary at all upon the removal of ATP. The rigor state was confirmed by stiffness measurements made after small-amplitude (0.75%), quick length changes. Upon re-relaxation, the stiffness of the skinned fiber decreased to the value of the resting state (4 mM ATP) and the phase angle delta returned to its original value. A model based on either anisotropic subunit-2 (S-2) movements or other cross-bridge-related structural anisotropy (form birefringence) changes during the relaxed-rigor transition is suggested.

Calcium transport, ATPase activity and lipid composition in sarcoplasmic reticulum isolated from isogenic lines of normal and dystrophic chickens.

Two new lines of chickens with near identical genotypes (greater than 90% isogeneity), one demonstrating avian dystrophy, were used for isolation of sarcoplasmic reticulum vesicles. Vesicles from line 433 (dystrophic) displayed reduced Ca2+-ATPase activity, phosphoenzyme formation and steady-state calcium transport capabilities in comparison with vesicles from line 03 (normal). Lipid analyses show that dystrophic vesicles have greater amounts of cholesterol and lesser amounts of phosphatidylcholine. The results support the use of isogenic chickens in further studies of avian dystrophy. However, the results also suggest that current sarcoplasmic reticulum vesicle purification procedures dependent on differential calcium accumulation may not fully achieve the intended purpose.