Comparison of Outcomes Following a Switch From a Brand to an Authorized Versus Independent Generic Drug.

Authorized generics are identical in formulation to brand drugs, manufactured by the brand company but marketed as a generic. Generics, marketed by generic manufacturers, are required to demonstrate pharmaceutical and bioequivalence to the brand drug, but repetition of clinical trials is not required. This retrospective cohort study compared outcomes for generics and authorized generics, which serves as a generic vs. brand proxy that minimizes bias against generics. For the seven drugs studied between 1999 and 2014, 5,234 unique patients were on brand drugs prior to generic entry and 4,900 (93.6%) switched to a generic. During the 12 months following the brand-to-generic switch, patients using generics vs. authorized generics were similar in terms of outpatient visits, urgent care visits, hospitalizations, and medication discontinuation. The likelihood of emergency department (ED) visits was slightly higher for authorized generics compared with generics. These data suggest that generics were clinically no worse than their proxy brand comparators.

Steady dynein forces induce flutter instability and propagating waves in mathematical models of flagella.

Cilia and flagella are highly conserved organelles that beat rhythmically with propulsive, oscillatory waveforms. The mechanism that produces these autonomous oscillations remains a mystery. It is widely believed that dynein activity must be dynamically regulated (switched on and off, or modulated) on opposite sides of the axoneme to produce oscillations. A variety of regulation mechanisms have been proposed based on feedback from mechanical deformation to dynein force. In this paper, we show that a much simpler interaction between dynein and the passive components of the axoneme can produce coordinated, propulsive oscillations. Steady, distributed axial forces, acting in opposite directions on coupled beams in viscous fluid, lead to dynamic structural instability and oscillatory, wave-like motion. This 'flutter' instability is a dynamic analogue to the well-known static instability, buckling. Flutter also occurs in slender beams subjected to tangential axial loads, in aircraft wings exposed to steady air flow and in flexible pipes conveying fluid. By analysis of the flagellar equations of motion and simulation of structural models of flagella, we demonstrate that dynein does not need to switch direction or inactivate to produce autonomous, propulsive oscillations, but must simply pull steadily above a critical threshold force.

Propulsive forces on the flagellum during locomotion of Chlamydomonas reinhardtii.

The distributed propulsive forces exerted on the flagellum of the swimming alga Chlamydomonas reinhardtii by surrounding fluid were estimated from experimental image data. Images of uniflagellate mutant Chlamydomonas cells were obtained at 350 frames/s with 125-nm spatial resolution, and the motion of the cell body and the flagellum were analyzed in the context of low-Reynolds-number fluid mechanics. Wild-type uniflagellate cells, as well as uniflagellate cells lacking inner dynein arms (ida3) or outer dynein arms (oda2) were studied. Ida3 cells exhibit stunted flagellar waveforms, whereas oda2 cells beat with lower frequency. Image registration and sorting algorithms provided high-resolution estimates of the motion of the cell body, as well as detailed kinematics of the flagellum. The swimming cell was modeled as an ellipsoid in Stokes flow, propelled by viscous forces on the flagellum. The normal and tangential components of force on the flagellum (f(N) and f(T)) were related by resistive coefficients (C(N) and C(T)) to the corresponding components of velocity (V(N) and V(T)).The values of these coefficients were estimated by satisfying equilibrium requirements for force and torque on the cell. The estimated values of the resistive coefficients are consistent among all three genotypes and similar to theoretical predictions.

Efficient spatiotemporal analysis of the flagellar waveform of Chlamydomonas reinhardtii.

The 9 + 2 axoneme is a microtubule-based machine that powers the oscillatory beating of cilia and flagella. Its highly regulated movement is essential for the normal function of many organs; ciliopathies cause congenital defects, chronic respiratory tract infections and infertility. We present an efficient method to obtain a quantitative description of flagellar motion, with high spatial and temporal resolution, from high speed video recording of bright field images. This highly automated technique provides the shape, shear angle, curvature, and bend propagation speeds along the length of the flagellum, with approximately 200 temporal samples per beat. We compared the waveforms of uniflagellated wild-type and ida3 mutant cells, which lack the I1 inner dynein complex. Video images were captured at 350 fps. Rigid-body motion was eliminated by fast Fourier transform (FFT)-based registration, and the Cartesian (x-y) coordinates of points on the flagellum were identified. These x-y "point clouds" were embedded in two data dimensions using Isomap, a nonlinear dimension reduction method, and sorted by phase in the flagellar cycle. A smooth surface was fitted to the sorted point clouds, which provides high-resolution estimates of shear angle and curvature. Wild-type and ida3 cells exhibit large differences in shear amplitude, but similar maximum and minimum curvature values. In ida3 cells, the reverse bend begins earlier and travels more slowly relative to the principal bend, than in wild-type cells. The regulation of flagellar movement must involve I1 dynein in a manner consistent with these results.

Motile organelles: the importance of specific tubulin isoforms.

The requirements for building flagellar axonemes and centrioles are beginning to be uncovered. The carboxyl terminus of a specific beta tubulin isoform plays an important role in forming the '9 + 2' structure of the axoneme; delta tubulin plays an essential role in forming the triplet microtubules of centrioles and basal bodies.

Gene expression in neurotrauma.

It has been established that following injury to the central nervous system two types of damage take place, the initial insult and the secondary response to injury. This review will focus on the secondary molecular aspects of neurotrauma. These responses may be either deleterious or have protective effects upon the injured cell population. Molecular responses include the regulation of genes which change cellular architecture, up-regulate of growth factors, induce reparative stress responses, influence apoptosis and regulate the transcriptional process. The purpose of this study is to provide the reader with a brief overview of some of the molecular mechanisms which are activated following a neurological insult.

Craniocerebral missile injuries.

Gun shot wounds to the brain are among the most devastating causes of morbidity and mortality in the civilian population. The majority of the victims will not survive and for a great number of survivors life becomes an uphill battle with permanent deficits and complications. While the fundamental surgical care of these patients is essentially unchanged, our scientific understanding of the pathophysiological changes and the post-injury care of the victims has been evolving. The purpose of this article is to provide an overview of the current clinical and laboratory advances in understanding and treating gun shot injuries to the brain.

The tubulin fraternity: alpha to eta.

Microtubules perform essential and diverse functions in eukaryotic cells. They are required for spindles during mitosis and meiosis, for axonal transport, for organelle positioning, and for cilia and flagella. gamma-tubulin is necessary to initiate the assembly of alpha-beta tubulin heterodimers into microtubule polymers. Recent genetic analyses and database searches have added four new members of the tubulin superfamily, which now includes alpha-, beta-, gamma-, delta-, epsilon-, zeta-, and eta-tubulin.

Extragenic bypass suppressors of mutations in the essential gene BLD2 promote assembly of basal bodies with abnormal microtubules in Chlamydomonas reinhardtii.

bld2-1 mutant Chlamydomonas reinhardtii strains assemble basal bodies with singlet microtubules; bld2-1 cells display flagellar assembly defects as well as positioning defects of the mitotic spindle and cleavage furrow. To further understand the role of the BLD2 gene, we have isolated three new bld2 alleles and three partially dominant extragenic suppressors, rgn1-1, rgn1-2, and rgn1-3. bld2 rgn1-1 strains have phenotypes intermediate between those of bld2 and wild-type strains with respect to flagellar number, microtubule rootlet organization, cleavage furrow positioning, and basal body structural phenotypes. Instead of the triplet microtubules of wild-type cells, bld2 rgn1-1 basal bodies have mixtures of no, singlet, doublet, and triplet microtubules. The bld2-4 allele was made by insertional mutagenesis and identified in a noncomplementation screen in a diploid strain. The bld2-4 allele has a lethal phenotype based on mitotic segregation in diploid strains and in haploid strains generated by meiotic recombination. The lethal phenotype in haploid strains is suppressed by rgn1-1; these suppressed strains have similar phenotypes to other bld2 rgn1-1 double mutants. It is likely that BLD2 is an essential gene that is needed for basal body assembly and function.

Chlamydomonas reinhardtii: biological rationale for genomics.

Chlamydomonas reinhardtii has been the subject of genetic, biochemical, cytological, and molecular analyses for over 50 years. It is an ideal model system for the study of flagella and basal bodies as well as the study of photosynthesis and chloroplast biogenesis, cell-cell recognition and fusion, phototaxis, and secretion. It is clear that many of the genes identified in Chlamydomonas have homologs in land plants as well as animals. Thus, a genomic approach in Chlamydomonas will provide another important avenue for the understanding of important biological processes.

Eyespot-assembly mutants in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.

Patterns of immediate early gene mRNA expression following rodent and human traumatic brain injury.

Cell stimulation which leads to degeneration triggers a prolonged wave of immediate early gene (IEG) transcription that correlates with neuronal demise. In order to determine the relevance of the prolonged IEG response to human traumatic brain injury, we analyzed IEG mRNA levels in brain tissue isolated following a controlled penetrating injury and an injection of the excitotoxin Quinolinic acid (QA), as well as from tissue recovered during routine neurosurgery for trauma. Total RNA was extracted from tissue and subjected to Northern analysis of IEG mRNAs (c-fos and zif/268). Both models produced rapid and prolonged waves of IEG transcription that appeared to correlate with the severity of injury. Increases in zif/268 mRNA were observed within 1 h with levels reaching their peak at 6 h following excitotoxic injury and 3 h following a controlled penetration. In general, human traumatic brain injury resulted in variable increases in IEG mRNA levels following traumatic injury with the largest IEG mRNA increases observed in tissue collected 0-10 h after injury. This post-injury time corresponds to the peak of the prolonged IEG response observed in rodents following excitotoxic injury. Comparisons were made in IEG response between rodent frontal cortex and human cortex, because the majority of the human tissue originated from the cerebral cortex. These results further support the hypothesis that prolonged IEG transcription serves as a marker of traumatic brain injury and may play a role in neurodegeneration and/or glial activation. Moreover, observations of similar IEG patterns of expression reinforces the importance of rodent models of brain injury providing useful information directly applicable to human brain injury.

Patterns of heat-shock protein 70 biosynthesis following human traumatic brain injury.

Heat-shock protein 70 (hsp70) is activated upon cellular stress/injury and participates in the folding and intracellular transport of damaged proteins. The expression of hsp70 following CNS trauma has been speculated to be part of a cellular response which is involved in the repair of damaged proteins. In this study, we measured hsp70 mRNA and protein levels within human cerebral cortex subjected to traumatic brain injury. Specimens were obtained during routine neurosurgery for trauma and processed for Northern mRNA and Western protein analysis. The largest increase in hsp70 mRNA levels was detected in trauma tissue obtained 4-6 h following injury. By 24 h, hsp70 mRNA levels were similar to nontrauma comparison tissues. hsp70 protein expression exhibited its greatest increases at 12-20 h post-injury. Immunocytological techniques revealed hsp70 protein expression in cells with neuronal-like morphology at 12 h after injury. These results suggest a role for hsp70 in human cortex following TBI. Moreover, since the temporal induction pattern of hsp70 biosynthesis is similar to that reported in the rodent, our observations validate the importance of rodent brain injury models in providing useful information directly applicable to human brain injury.

Heat-shock protein 72 expression in excitotoxic versus penetrating injuries of the rodent cerebral cortex.

The induction of heat shock protein 72 (hsp72) has been described in various experimental models of brain injury. The present study examined hsp72 expression patterns within the rodent cerebral cortex in experimental paradigms designed to mimic two mechanisms of damage produced by penetration of the cerebral cortex: (1) tissue tearing from the missile track and (2) diffuse excitotoxicity during temporary cavitation and shock wave formation. Adult male Spaque-Dawley rats received controlled penetration (stab) or injection of the NMDA receptor excitotoxin, quinolinic acid (QA), into the frontal cortex and were killed 1-24 h later. Tissue from the lesioned, sham-operated, or contralateral uninjected cortex was processed for Western and immunocytochemical analyses of hsp72 protein expression. By 12 h, both controlled penetration and excitotoxic brain injuries produced significant increases in hsp72 immunoreactivity, which decreased toward control levels at 24 h. However, the severity and regional distribution of hsp72 expression varied between the two models. Specifically, the controlled penetration injury produced many hsp72-expressing cells near the needle track, while immunoreactive cells within the QA-injected cortex were found in the periphery of the lesion site. Morphological assessment of brain sections subjected to dual-labeling procedures demonstrated that cells expressing hsp72 were primarily neuronal in both models of injury. These results suggest that although controlled penetration and diffuse excitotoxicity may induce similar temporal and cellular patterns of hsp72 expression, the spatial location of hsp72-immunoreactive cells may differ between the two models.

Identification of the gene encoding the tryptophan synthase beta-subunit from Chlamydomonas reinhardtii.

We report the isolation of a Chlamydomonas reinhardtii cDNA that encodes the beta-subunit of tryptophan synthase (TSB). This cDNA was cloned by functional complementation of a trp-operon-deleted strain of Escherichia coli. Hybridization analysis indicated that the gene exists in a single copy. The predicted amino acid sequence showed the greatest identity to TSB polypeptides from other photosynthetic organisms. With the goal of identifying mutations in the gene encoding this enzyme, we isolated 11 recessive and 1 dominant single-gene mutation that conferred resistance to 5-fluoroindole. These mutations fell into three complementation groups, MAA2, MAA7, and TAR1. In vitro assays showed that mutations at each of these loci affected TSB activity. Restriction fragment-length polymorphism analysis suggested that MAA7 encodes TSB. MAA2 and TAR1 may act to regulate the activity of MAA7 or its protein product.

Pharmacological and genetic evidence for a role of rootlet and phycoplast microtubules in the positioning and assembly of cleavage furrows in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, specialized cytoskeletal structures known as rootlet microtubules are present throughout interphase and mitosis. During cytokinesis, an array of microtubules termed the phycoplast is nucleated from rootlet microtubules and forms coincidentally with the cleavage furrow [Johnson and Porter, 1968: J. Cell Biol. 38:403-425; Holmes and Dutcher, 1989: J. Cell Sci. 94:273-285; Gaffel and el-Gammel, 1990: Protoplasma 156:139-148; Schibler and Huang, 1991: J. Cell Biol. 113:605-614]. We have obtained two independent lines of evidence that support the hypothesis that the rootlet and phycoplast microtubules play a direct role in cleavage furrow placement and assembly. First, the destabilization of spindle and phycoplast microtubules by pharmacological agents was accompanied by the aberrant distribution of actin and a failure of cytokinesis. Second, we characterized mutant strains that failed to complete cytokinesis properly. Actin and myosin were mislocalized to additional rootlet microtubules in the cyt2-1 strain, and this mislocalization was correlated with the presence of additional cleavage furrows. This evidence suggests that microtubules are necessary for the correct positioning and assembly of functional cleavage furrows in C. reinhardtii.

The UNI3 gene is required for assembly of basal bodies of Chlamydomonas and encodes delta-tubulin, a new member of the tubulin superfamily.

We have cloned the UNI3 gene in Chlamydomonas and find that it encodes a new member of the tubulin superfamily. Although Uni3p shares significant sequence identity with alpha-, beta-, and gamma-tubulins, there is a region of Uni3p that has no similarity to tubulins or other known proteins. Mutant uni3-1 cells assemble zero, one, or two flagella. Pedigree analysis suggests that flagellar number in uni3-1 cells is a function of the age of the cell. The uniflagellate uni3-1 cells show a positional phenotype; the basal body opposite the eyespot templates the single flagellum. A percentage of uni3-1 cells also fail to orient the cleavage furrow properly, and basal bodies have been implicated in the placement of cleavage furrows in Chlamydomonas. Finally when uni3-1 cells are observed by electron microscopy, doublet rather than triplet microtubules are observed at the proximal end of the basal bodies. We propose that the Uni3 tubulin is involved in both the function and cell cycle-dependent maturation of basal bodies/centrioles.

Phosphoregulation of an inner dynein arm complex in Chlamydomonas reinhardtii is altered in phototactic mutant strains.

To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning.

Loss of spatial control of the mitotic spindle apparatus in a Chlamydomonas reinhardtii mutant strain lacking basal bodies.

The bld2-1 mutation in the green alga Chlamydomonas reinhardtii is the only known mutation that results in the loss of centrioles/basal bodies and the loss of coordination between spindle position and cleavage furrow position during cell division. Based on several different assays, bld2-1 cells lack basal bodies in > 99% of cells. The stereotypical cytoskeletal morphology and precise positioning of the cleavage furrow observed in wild-type cells is disrupted in bld2-1 cells. The positions of the mitotic spindle and of the cleavage furrow are not correlated with respect to each other or with a specific cellular landmark during cell division in bld2-1 cells. Actin has a variable distribution during mitosis in bld2-1 cells, but this aberrant distribution is not correlated with the spindle positioning defect. In both wild-type and bld2-1 cells, the position of the cleavage furrow is coincident with a specialized set of microtubules found in green algae known as the rootlet microtubules. We propose that the rootlet microtubules perform the functions of astral microtubules and that functional centrioles are necessary for the organization of the cytoskeletal superstructure critical for correct spindle and cleavage furrow placement in Chlamydomonas.