Exercise, manual therapy, and use of booster sessions in physical therapy for knee osteoarthritis: a multi-center, factorial randomized clinical trial.

OBJECTIVE: (1) Do treatment effects differ between participants receiving manual therapy (MT) with exercise compared to subjects who don't, (2) are treatment effects sustained better when participants receive booster sessions compared to those who don't over a one year period in subjects with knee osteoarthritis (KOA)? DESIGN: Multi-center, 2 × 2 factorial randomized clinical trial. 300 participants with knee OA were randomized to four groups: exercise-no boosters (Ex), exercise-with boosters (Ex+B), manual therapy+exercise-no boosters (MT+Ex), manual therapy+exercise-with boosters (MT+Ex+B). The primary outcome was the Western Ontario and McMaster osteoarthritis index (WOMAC) at 1 year. Secondary outcomes included knee pain, physical performance tests, and proportions of participants meeting treatment responder criteria. RESULTS: There were no differences between groups on the WOMAC at 1 year or on any performance-based measures. Secondary analyses indicated a) better scores on the WOMAC and greater odds of being a treatment responder at 9 weeks for participants receiving MT, b) greater odds of being a treatment responder at 1 year for participants receiving boosters. Exploratory interaction analysis suggested knee pain decreases for participants receiving boosters and increases for participants not receiving boosters from 9 weeks to 1 year. CONCLUSIONS: MT or use of boosters with exercise did not result in additive improvement in the primary outcome at 1 year. Secondary outcomes suggest MT may have some short term benefit, and booster sessions may improve responder status and knee pain at 1 year. However, the role of booster sessions remains unclear in sustaining treatment effects and warrants further study. CLINICAL TRIALS: gov (NCT01314183).

Side-to-side weight-bearing asymmetry in subjects with low back pain.

The purpose of this project was to determine if subjects with low back pain (LBP) exhibit greater side-to-side weight-bearing (WB) asymmetry compared to healthy control subjects without LBP. This study utilized an observational double cohort design and consisted of 35 subjects with LBP and 31 healthy control subjects. Side-to-side WB asymmetry was calculated as the average of the absolute value of the difference between the right and left lower extremity from three trials. The percentage of the average side-to-side WB asymmetry relative to the total body weight was calculated to normalize expected differences in magnitude of asymmetry based on a subject's total body weight. An 11-point numeric pain rating scale was used to represent the subject's current level of pain. Patients with LBP demonstrated significantly greater normalized side-to-side WB asymmetry than healthy control subjects (8.8% vs. 3.6%, respectively, P<0.001). In patients with LBP, higher magnitudes of side-to-side WB asymmetry were significantly associated with increased pain (r=0.39, P=0.021). In conclusion patients with LBP exhibited increased side-to-side WB asymmetry compared to healthy control subjects without LBP. This asymmetry was associated with increased levels of pain. This finding is relevant for planning future studies that will attempt to provide evidence for the construct validity of manipulation by determining if side-to-side WB asymmetry normalizes after a manipulation intervention and if this improvement is associated with improvements in pain and function.

A flavin reductase stimulates DszA and DszC proteins of Rhodococcus erythropolis IGTS8 in vitro.

Rhodococcus erythropolis IGTS8 is a gram positive bacterium, which can catabolize dibenzothiophene to 2-hydroxybiphenyl and inorganic sulfur without the cleavage of carbon-carbon bonds. Three structural genes, dszA, dszB, and dszC, have been cloned and shown to be necessary for this phenotype. Here, we demonstrate that a FMN:NADPH oxidoreductase from Vibrio harveyi complements activities of purified DszA and DszC proteins. Furthermore, we propose that DszA and DszC are oxygenase units that do not use NAD(P)H directly, but instead use FMNH2 from a FMN:NADPH oxidoreductase for oxygenation.

Genetic analysis of the dsz promoter and associated regulatory regions of Rhodococcus erythropolis IGTS8.

The dsz gene cluster of Rhodococcus erythropolis IGTS8 comprises three genes, dszA, dszB, and dszC, whose products are involved in the conversion of dibenzothiophene (DBT) to 2-hydroxybiphenyl and sulfite. This organism can use DBT as the sole sulfur source but not as a carbon source. Dsz activity is repressed by methionine, cysteine, Casamino Acids, and sulfate but not by DBT or dimethyl sulfoxide. We cloned 385 bp of the DNA immediately 5' to dszA in front of the reporter gene lacZ of Escherichia coli. We showed that this region contains a Rhodococcus promoter and at least three dsz regulatory regions. After hydrazine mutagenesis of this DNA, colonies that were able to express beta-galactosidase in the presence of Casamino Acids were isolated. Sequencing of these mutants revealed two possible regulatory regions. One is at -263 to -244, and the other is at -93 to -38, where -1 is the base preceding the A of the initiation codon ATG of dszA. An S1 nuclease protection assay showed that the start of the dsz promoter is the G at -46 and that transcription is repressed by sulfate and cysteine but not by dimethyl sulfoxide. The promoter encompasses a region of potential diad symmetry that may contain an operator. Immediately upstream of the promoter is a protein-binding domain between -146 and -121. Deletion of this region did not affect repression, but promoter activity appeared to be reduced by threefold. Thus, it could be an activator binding site or an enhancer region.

Mapping receptor binding sites in interleukin (IL)-1 receptor antagonist and IL-1 beta by site-directed mutagenesis. Identification of a single site in IL-1ra and two sites in IL-1 beta.

Interleukin-1 receptor antagonist (IL-1ra), an IL-1 family member, binds with high affinity to the type I IL-1 receptor (IL-1RI), blocking IL-1 binding but not inducing an IL-1-like response. Extensive site-directed mutagenesis has been used to identify residues in IL-1ra and IL-1 beta involved in binding to IL-1RI. These analyses have revealed the presence of two discrete receptor binding sites on IL-1 beta. Only one of these sites is present on IL-1ra, consisting of residues Trp-16, Gln-20, Tyr-34, Gln-36, and Tyr-147. Interestingly, the absent second site is at the location of the major structural difference between IL-1ra and IL-1 beta, which are otherwise structurally similar. The two receptor binding sites on IL-1 beta are also present on IL-1 alpha. Thus, it appears that the two IL-1 agonist molecules have two sites for IL-1RI binding, and the homologous antagonist molecule, IL-1ra, has only one. Based on these observations, a hypothesis is presented to account for the difference in activity between the agonist and antagonist proteins. It is proposed that the presence of the two receptor binding sites may be necessary for agonist activity.

Evidence that bacteriophage T4 eph1 is a missense hoc mutation.

An electrophoretic mutation of bacteriophage T4, eph1, appears to code for a missense hoc (highly antigenic outer capsid) protein. This is based on the observation that particles lacking hoc protein (hoc- particles), after incubation in a crude extract of Escherichia coli infected with phage carrying the eph1 mutation acquired the electrophoretic mobility of the eph1 strain (the electrophoretic mobility of the eph1 strain itself is slower than that of hoc- particles). Thus, it is likely that during infection of E. coli with the eph1 strain, a hoc protein is made that has a lower negative charge than normal hoc protein but can nevertheless bind to particles lacking hoc protein. These results confirm that eph1 is a hoc mutation.

Pyrimidine dimers induced in Escherichia coli DNA by ultraviolet radiation present in sunlight.

Escherichia coli DNA was irradiated with various wavelengths of monochromatic UV light from 254 to 320 nm, and the relative yields of the different cyclobutane pyrimidine dimers determined. Cytosine-thymine dimers (C mean value of T) were more frequent than thymine dimers (T mean value of T) at low fluences of 300 and 313 nm light, whereas the reverse was true at either longer or shorter wavelengths. Thus, in the solar UV range deemed responsible for skin cancer (i.e. 295-315 nm), C mean value of T are probably more important than T mean value of T.

The effect of a change in mutation rate on the incidence of dominant and X-linked recessive disorders in man.

In order to assess the impact on man of a sustained change in mutation rate that might be caused by ionizing radiation or a chemical mutagen in the environment, it is important to determine the current incidence of genetic disease, the rate at which deleterious mutations arise and the number of generations that mutations persist before eliminated by selection. From these data it should be possible to estimate both the increase in genetic disease in the first generation following the increase in mutation rate, and the rate at which a new equilibrium between mutation and selection would occur. In this paper the results of a survey to determine birth frequency, mutation rate and reproductive fitness for each of the important dominant and X-linked recessive disorders are described. It is estimated that these disorders affect about 0.6% of live-born individuals, including 0.1% of live-borns who carry a newly-arising mutation. These figures are approx. 50% lower than those used by the various committees that have assessed the genetic risk to man from ionizing radiation. If the mutation rate were to permanently double, the frequency of these disorders would be expected in increase in the first generation by 15%, to 0.7% of live-births. The increase in the first 2 generations would be 24% and a 50% increase would occur by the 9th generation. A calculation of the possible increase in dominant and X-linked recessive disorders due to exposure of a population to ionizing radiation indicates that the estimate made in 1977 by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) may be too high by a factor of 2-6 fold.

Isolation and genetic properties of a bacteriophage T4 uvsX mutant.

A technique for isolating UV-sensitive mutants of bacteriophage T4 and a relatively simple method for localizing the UV-sensitive mutations on the genetic map are described. Of 13 mutants isolated 5 were shown to be den V mutants and 1 was shown to be a uvsX mutant. Of the remainder, 4 are probably uvsX mutants (based on their map location) while the indentity of the remaining 3 has not been determined. The uvsX mutant (uvsX102) had enhanced UV and gamma-ray sensitivity compared to the only other well characterized mutant in this gene, uvsX1 (T4x). Both uvsX1 and uvxX102 were more UV-sensitivity when plated on the su- E. coli B hosts, B and S/6, compared to the su+ K12 host CR63, and both reduced the frequency of recombination to the same extent (2--3-fold). The uvsX gene is located between gene 41 and beta gt.

Evidence for a near UV-induced photoproduct of 5-hydroxymethylcytosine in bacteriophage T4 that can be recognized by endonuclease V.

Non-photoreactivable endonuclease V-sensitive sites have been detected in the DNA of wild type bacteriophage T4 irradiated with near UV light (320 nm). Such sites were not detected in the DNA of (a) wild type T4 irradiated with far UV (254 nm) or (B) in T4 mutants in which non-glucosylated 5-hydroxy-methylcytosine (5HMC) or cytosine replaces glucosylated 5HMC normally present in T4, irradiated with 320 nm or 254 nm light. Although the non-photoreactivable sites accounted for approximately 50% of the endonuclease V-sensitive sites in the DNA of glucosylated T4 irradiated with near UV, there was very little difference in the sensitivities of T4 containing glucosylated 5HMC, non-glucosylated 5HMC and cytosine to near UV (313 nm). We propose that the photoproduct responsible for the non-photoreactivable, but endonuclease V-sensitive, sites in glucosylated DNA is formed from glucosylated 5HMC and that a similar photoproduct is formed from non-glucosylated 5HMC or cytosine in the appropriate phage strains. We further propose that the glucosylated 5HMC photoproduct is non-photoreactivable whereas the cytosine and non-glucosylated 5HMC photoproducts are photoreactivable and are therefore possibly cyclobutane dimers.

Conditional lethal mutants of bacteriophage T4 unable to grow on a streptomycin resistant mutant of Escherichia coli.

Sixteen conditional lethal mutants of bacteriophage T4D have been isolated which grow on Escherichia coli CR63 (a su+ streptomycin-sensitive K12 strain) but are restricted by CR/s (a streptomycin-resistant derivative of CR63). These mutants have been given the prefix str. Four of these mutants are amber and 12 appear to be missense. Eleven of the 12 missense mutants appear to be "pseudo-amber" (i.e. they are restricted by a su- E. coli B strain but not by a su- K12 strain); the other missense mutant was not restricted by either B or K12. The str mutations mapped in 12 different genes. Most were clustered in a region of early genes (gene 56 to gene 47). Fifty-eight amber and 10 "pseudo-amber" mutants isolated previously for their inability to grow on E. coli B were tested for restriction by CR/s. All the amber mutants grew normally on CR/s, whereas all 10 "pseudo-amber" mutants were restricted by CR/s. This implies that the phenotype of the "pseudo-amber" mutants is the result of a ribosomal difference between the permissive host CR63 and the restrictive hosts B and CR/s. These str mutants should prove to be useful alternatives to amber mutants for genetic and biochemical studies of bacteriophage T4 and for studies of the E. coli ribosome. It should be possible ot isolate similar mutants in other bacteriophages provided that streptomycin resistant hosts are available.

Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles.

A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses.

Superinfection exclusion by incomplete genomes of bacteriophage T4.

The genetic basis of superinfection exclusion by bacteriophage T4 was investigated by using incomplete genomes derived from the gene 66 mutant E920g. Incomplete genomes, which included a region of T4 between genes 42 and 44, were able to exclude superinfecting phage with an efficiency similar to that of complete genomes. Those genomes which did not include this region were unable to exclude superinfecting phage. A mutant with reduced ability to exclude super-infecting phage was isolated after mutagenesis with hydroxylamine. The mutation maps midway between amN122 in gene 42 and amB22 in gene 43. The efficiency of exclusion of superinfecting phage (as measured by the percentage of superinfected cells which failed to release any phage carrying selected markers of the superinfecting phage) by this mutant was 50 to 60%, whereas for wild type it was 85 to 95%. Uptake of (3)H-leucine by cells infected with the mutant was inhibited by superinfection with ghosts and it has therefore been designated imm1, for lack of immunity to superinfecting phage and ghosts. The formation of infective centers by cells infected with imm1 or another imm(-) mutant (imm2) was not inhibited by superinfection with ghosts.

Identification and genetic characterization of mutants of bacteriophage T4 defective in the ability to induce exonuclease A.

A mutant of bacteriophage T4 unable to induce exonuclease A has been isolated. The mutation responsible for this defect maps between genes 39 and 56, in a region of the chromosome devoid of other known markers. Four deletion mutants lacking part of the genome located between genes 39 and 56 also fail to induce exonuclease A. The ability of all of these mutants to replicate suggests that exonuclease A is not essential for replication of phage T4.