The influence of sagittal center of pressure offset on gait kinematics and kinetics

ObjectivesKinetic patterns of the lower extremity joints have been shown to be influenced by modification of the location of the center of pressure (CoP) of the foot. The accepted theory is that a shifted location of the CoP alters the distance between the ground reaction force and the center of the joint, thereby modifying torques during gait. Various footwear designs have been reported to significantly alter the magnitude of sagittal joint torques during gait. However, the relationship between the CoP and the kinetic patterns in the sagittal plane has not been examined. The aim of this study was to evaluate the association between the sagittal location of the CoP and gait patterns during gait in healthy men.MethodsA foot-worn biomechanical device which allows controlled manipulation of the CoP location was utilized. Fourteen healthy men underwent successive gait analysis with the device set to convey three different sagittal locations of the CoP: neutral, anterior offset and posterior offset.ResultsCoP translation in the sagittal plane (i.e., from posterior to anterior) significantly related with an ankle dorsiflexion torque and a knee extension torque shift throughout the stance phase. Likewise, an anterior translation of the CoP significantly reduced the extension torque at the hip during pre-swing.ConclusionsThe study results confirm a direct correlation between sagittal offset of the CoP and the magnitude of joint torques throughout the lower extremity.     

The DR1 and DR6 First Exons of Human Herpesvirus 6A Are Not Required for Virus Replication in Culture and Are Deleted in Virus Stocks That Replicate Well in T-Cell Lines

Human herpesvirus 6A (HHV-6A) and HHV-6B are lymphotropic viruses which replicate in cultured activated cord blood mononuclear cells (CBMCs) and in T-cell lines. Viral genomes are composed of 143-kb unique (U) sequences flanked by ∼8- to 10-kb left and right direct repeats, DR_L and DR_R. We have recently cloned HHV-6A (U1102) into bacterial artificial chromosome (BAC) vectors, employing DNA replicative intermediates. Surprisingly, HHV-6A BACs and their parental DNAs were found to contain short ∼2.7-kb DRs. To test whether DR shortening occurred during passaging in CBMCs or in the SupT1 T-cell line, we compared packaged DNAs from various passages. Restriction enzymes, PCR, and sequencing analyses have shown the following. (i) Early (1992) viral preparations from CBMCs contained ∼8-kb DRs. (ii) Viruses currently propagated in SupT1 cells contained ∼2.7-kb DRs. (iii) The deletion spans positions 60 to 5545 in DR_L, including genes encoded by DR1 through the first exon of DR6. The pac-2-pac-1 packaging signals, the DR7 open reading frame (ORF), and the DR6 second exon were not deleted. (iv) The DR_R sequence was similarly shortened by 5.4 kb. (v) The DR1 through DR6 first exon sequences were deleted from the entire HHV-6A BACs, revealing that they were not translocated into other genome locations. (vi) When virus initially cultured in CBMCs was passaged in SupT1 cells no DR shortening occurred. (vii) Viral stocks possessing short DRs replicated efficiently, revealing the plasticity of herpesvirus genomes. We conclude that the DR deletion occurred once, producing virus with advantageous growth “conquering” the population. The DR1 gene and the first DR6 exon are not required for propagation in culture.Human herpesvirus 6 (HHV-6) is a member of the Betaherpesvirus subfamily, as recently reviewed (46). The virus can enter hematopoietic cells, including T cells, B cells, natural killer (NK) cells, monocytes, and dendritic cells (DCs), as well as nonhematopoietic cells, as reviewed in references 8, 17, and 46. In culture, the virus replicates in activated peripheral blood lymphocytes (PBLs), cord blood mononuclear cells (CBMCs), and in T-cell lines (1, 17, 46). HHV-6 isolates fall into two distinct classes designated as HHV-6A and HHV-6B variants. The two variants can be distinguished by their restriction enzyme patterns, antigenicity, DNA sequences, and disease association (1, 36, 46). HHV-6B is the causative agent of roseola infantum, a prevalent children''s disease characterized by high fever and skin rash (47). In rare cases, the virus exhibits neurotropism and has been found in children experiencing convulsions up to lethal encephalitis (1, 21, 46, 48).HHV-6B reactivation from latency was found to occur in patients receiving immunosuppressive treatment in bone marrow and other transplantations. This was associated with febrile illness, delayed transplant engraftment, and neurological involvement, up to lethal encephalitis (5, 13, 34, 46). HHV-6A has thus far no clear disease association, although several studies have suggested central nervous system (CNS) tropism, including aggravation of symptoms in patients with multiple sclerosis (MS) (6, 14, 33, 41).HHV-6A and HHV-6B share general genomic architecture. The unit-length DNA molecules are approximately 160 kb, composed of a 143-kb unique (U) segment flanked by left and right direct repeats (DR_L and DR_R, respectively) (19, 24, 27, 46). The DRs are of sizes 8 to 10 kb in different viral isolates (2, 19, 24, 46). In both the HHV-6A and HHV-6B genomes, the herpesvirus conserved cleavage/packaging signals pac-1 and pac-2 (9, 15, 17) are located at the left and the right termini of the DRs (17, 19, 46). The PubMed sequence for the U1102 strain (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_001664","term_id":"1344462938","term_text":"NC_001664"}}NC_001664) starts with the pac-1 signal at positions 1 to 56, followed by multiple copies of perfect and imperfect telomere-like sequences, up to position 418. It was suggested that the telomeric repeats may have originated from host cell chromosomal telomeres (43). Additionally, the DR encodes several open reading frames (ORFs), four of which are dealt with in our paper: (i) the spliced DR1 at positions 501 to 759 and 843 to 2653; (ii) DR5 at positions 3738 to 4164; (iii) the spliced DR6 at positions 4725 to 5028 and 5837 to 6720; and (iv) an ORF of DR7, at positions 5629 to 6720, partially overlapping the DR6 gene (20). Hollsberg and coworkers (37) have recently found that the homologous gene in HHV-6B encodes a nuclear protein that forms a complex with viral DNA processivity factor p41. Gompels and coworkers have also shown that DR1 and DR6 are partly homologous to the human cytomegalovirus (HCMV) US22 gene family. Both have a CXC motif: DR1 with homology to the HCMV US26 gene and DR6 with homology to the HCMV US22 gene (20). The map continues with reiterated perfect hexanucleotide telomeric sequences (GGGTAA)_n at positions 7655 to 8008 (19, 43). The number of telomeric repeats was found to vary in different viral strains (2, 43). The DR terminates with the pac-2 signal.We have recently cloned the intact HHV-6A genome into bacterial artificial chromosomes (BACs), by direct cloning of unit-length DNA produced from circular or head-to-tail replication intermediates into modified BAC vectors containing the green fluorescent protein (GFP) marker and ampicillin-puromycin (Amp-Puro) selection cassette (3). Surprisingly, the HHV-6A BAC clones as well as the parental HHV-6A (U1102) propagated in our laboratory in SupT1 cells were found to contain DRs of ∼2.7 kb instead of the expected ∼8- to 10-kb DRs, as in the early publications (19, 24, 27, 46) and in the PubMed sequence. This has raised the following questions. When did the deleted DRs arise? What was the detailed structure of deleted DRs?HHV-6 was discovered by Gallo and colleagues in 1986 (35), and viral isolates were obtained in multiple laboratories from AIDS patients, patients with lymphoproliferative disorders, and patients with roseola infantum (12, 26, 42, 45, 47). The isolates were propagated initially in activated PBLs and CBMCs and then in continuous T-cell lines, including HSB-2, J-JHAN, SupT1, Molt-3, and MT-4 (11, 45). The U1102 strain isolated by Downing and colleagues (12) was contributed to our laboratory by Robert Honess and was propagated first in activated PBLs and CBMCs (11, 18, 36, 45) and then in J-JHAN and SupT1 T cells (4, 30). To answer the question with regard to the origin of the short DRs and their structure, we have compared earlier viral HHV-6A passages with the currently propagated virus and the HHV-6A BAC clones. We describe here the detailed structure of the DR_L and DR_R of the “new” virus, containing the short ∼2.7-kb DR. We show that the deletion contained the left multiple repeats of telomere-like sequences and the ORFs from DR1 up to the DR6 first exon. Review of viral passaging since 1992 indicated that the deletion occurred spontaneously. The deleted viruses were stably and efficiently propagated in SupT1 T cells, indicating that the DR1 and DR6 first exons are not essential for virus in vitro replication.     

Mechanical reconfiguration mediates swallowing and rejection in Aplysia californica

Muscular hydrostats, such as tongues, trunks or tentacles, have fewer constraints on their degrees of freedom than musculoskeletal systems, so changes in a structure’s shape may alter the positions and lengths of other components (i.e., induce mechanical reconfiguration). We studied mechanical reconfiguration during rejection and swallowing in the marine mollusk Aplysia californica. During rejection, inedible material is pushed out of an animal’s buccal cavity. The grasper (radula/odontophore) closes on inedible material, and then a posterior muscle, I2, pushes the grasper toward the jaws (protracts it). After the material is released, an anterior muscle complex (the I1/I3/jaw complex) pushes the grasper toward the esophagus (retracts it). During swallowing, the grasper is protracted open, and then retracts closed, pulling in food. Grasper closure changes its shape. Magnetic resonance images show that grasper closure lengthens I2. A kinetic model quantified the changes in the ability of I2 and I1/I3 to exert force as grasper shape changed. Grasper closure increases I2’s ability to protract during rejection, and increases I1/I3’s ability to retract during swallowing. Motor neurons controlling radular closure may therefore affect the behavioral outputs of I2’s and I1/I3’s motor neurons. Thus, motor neurons may modulate the outputs of other motor neurons through mechanical reconfiguration.Valerie A. Novakovic and Gregory P. Sutton contributed equally to the paper.     

Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae)

Both Gazella gazella and Gazella dorcas are endangered species with continually dwindling population size, yet basic knowledge on their spermatozoa is missing. Semen collected post-mortem (PM) from the cauda epididymis of five adult gazelles (three Gazella gazella gazella, one Gazella gazella acaiae and one G. dorcas) was cryopreserved using directional freezing of large volumes (8 mL) with egg-yolk-free extender. Sperm size measurements and SYBR-14/propodium iodide (PI) viability stain validation for use in gazelles were conducted. Post-thaw characterization included motility, viability, acrosome damage evaluation, computerized motility characterization and morphology and sperm motility index (SMI) was calculated. Extracted sperm motility was 71.67+/-11.67% (mean+/-S.E.M.). Post-thaw motility ranged between 15% and 63%, viability was 57.49+/-3.24%, intact acrosome was detected in 63.74+/-2.6% (median 64.8%, upper/lower quartiles 71.79%, 61.82%), and normal morphology ranged between 41% and 63%. Motility characterization showed two sub-groups-highly active and progressively motile spermatozoa with SMI of 62.75+/-0.38 and low activity and poorly progressive with SMI of 46.16+/-1.53. Our results indicate that PM preservation of gazelle spermatozoa with satisfactory post-thaw viability is possible and cryobanking is achievable.     

Structural basis of DNA recognition by p53 tetramers

The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.     

Tracing the path of DNA substrates in active Tetrahymena telomerase holoenzyme complexes: mapping of DNA contact sites in the RNA subunit

Telomerase, the enzyme that extends single-stranded telomeric DNA, consists of an RNA subunit (TER) including a short template sequence, a catalytic protein (TERT) and accessory proteins. We used site-specific UV cross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA-DNA hybrids made with overlapping complementary oligodeoxynucleotides. These data allowed tracing of the DNA path through the telomerase complexes from the template to the TERT binding element (TBE) region of TER. TBE is known to bind TERT and to be involved in the template 5'-boundary definition. Based on these findings, we propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5'-end of the RNA template. The upstream DNA-TBE interaction may also function as an anchor for the subsequent realignment of the 3'-end of the DNA with the 3'-end of the template to enable initiation of synthesis of a new telomeric repeat.     

Reduction in knee adduction moment via non-invasive biomechanical training: a longitudinal gait analysis study

Biomechanical non-invasive interventions have been previously reported to reduce pain and facilitate superior levels of function in patients with medial knee osteoarthritis [OA]. One such treatment is the AposTherapy, a customized program utilizing a foot-worn biomechanical device allowing center of pressure modification and continuous perturbation during gait. The influence of this intervention on objective gait metrics has yet to be determined. The aim of the current study was to prospectively examine changes in kinetic and kinematic parameters in patients enrolled in this treatment program. Twenty-five females with symptomatic bilateral medial compartment knee OA were enrolled in the customized daily treatment program. All patients underwent barefoot gait analysis testing and completed subjective questionnaires prior to treatment initiation and on two follow-up visits. Significantly reduced knee adduction moment (KAM) magnitude was noted during barefoot walking after three and nine months of treatment. On average, the knee adduction impulse and the 1st and 2nd KAM peaks were reduced by 13%, 8.4%, and 12.7%, respectively. Furthermore, moment reduction was accompanied by elevated walking velocity, significant pain reduction, and increased functional activity. In addition to symptomatic improvement, our results suggest that this treatment program can alter kinetic gait parameters in this population. We speculate that these adaptations account for the symptomatic and functional improvement reported for this intervention.     

Photoperiodicity and increasing salinity as environmental cues for reproduction in desert adapted rodents

Understanding the ways environmental signals, regulate reproduction and reproductive behavior of desert adapted rodents is a major gap in our knowledge. In this study, we assessed the roles of photoperiod and diet salinity, as signals for reproduction. We challenged desert adapted common spiny mice, Acomys cahirinus, males and females with osmotic stress, by gradually increasing salinity in their water source - from 0.9% to 5% NaCl under short and long days (SD and LD, respectively). Photoperiodicity affected testosterone levels, as under LD-acclimation, levels were significantly (p<0.05) higher than under SD-acclimation. Salinity treatment (ST) significantly reduced SD-acclimated male body mass (W(b)) and testis mass (p<0.005; normalized to W(b)). ST-LD-females significantly (p<0.005) decreased progesterone levels and the numbers of estrous cycles. A reduction in white adipose tissue (WAT) to an undetectable level was noted in ST-mice of both sexes under both photoperiod regimes. Receptors for vasopressin (VP) and aldosterone were revealed on testes of all male groups and on WAT in control groups. Our results suggest that photoperiod serves as an initial signal while water availability, expressed by increased salinity in the water source, is an ultimate cue for regulation of reproduction, in both sexes of desert-adapted A. cahirinus. We assume that environmental changes also affect behavior, as water seeking behavior by selecting food items, or locomotor activity may change in extreme environment, and thus indirectly affect reproduction and reproductive behavior. The existence of VP and aldosterone receptors in the gonads and WAT suggests the involvement of osmoregulatory hormones in reproductive control of desert adapted rodents.     

Studies of Mg2+/Ca2+ complexes of naturally occurring dinucleotides: potentiometric titrations, NMR, and molecular dynamics

Dinucleotides (Np(n)N'; N and N' are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg(2+) and Ca(2+) complexes of Np(n)N's. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap(n)A-Mg(2+), n = 3, 4, 5, respectively). The base (A or G) or ion (Mg(2+) or Ca(2+)) has a minor effect on K(M)(ML) values. In a physiological medium, the longer Ap(n)As (n = 4, 5) are predicted to occur mostly as the Mg(2+)/Ca(2+) complexes. (31)P NMR monitored titrations of Np(n)N's with Mg(2+)/Ca(2+) ions showed that the middle phosphates of the dinucleotides coordinate with Mg(2+)/Ca(2+). Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap(2)A and Ap(4)A coordinate Mg(2+) and Ca(2+) ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np(n)N'-Mg(2+)/Ca(2+) complexes remains the same as that of free mononucleotides.     

Leaf-induced gibberellin signaling is essential for internode elongation, cambial activity, and fiber differentiation in tobacco stems

The gibberellins (GAs) are a group of endogenous compounds that promote the growth of most plant organs, including stem internodes. We show that in tobacco (Nicotiana tabacum) the presence of leaves is essential for the accumulation of bioactive GAs and their immediate precursors in the stem and consequently for normal stem elongation, cambial proliferation, and xylem fiber differentiation. These processes do not occur in the absence of maturing leaves but can be restored by application of C(19)-GAs, identifying the presence of leaves as a requirement for GA signaling in stems and revealing the fundamental role of GAs in secondary growth regulation. The use of reporter genes for GA activity and GA-directed DELLA protein degradation in Arabidopsis thaliana confirms the presence of a mobile signal from leaves to the stem that induces GA signaling.