Acute Postoperative Pain Due to Dental Extraction in the Adult Population: A Systematic Review and Network Meta-analysis.

This study compares the effectiveness of pharmacological treatments to develop guidelines for the management of acute pain after tooth extraction. We searched Medline, EMBASE, CENTRAL, and US Clinical Trials registry on November 21, 2020. We included randomized clinical trials (RCTs) of participants undergoing dental extractions comparing 10 interventions, including acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), opioids, and combinations to placebo. After duplicate screening and data abstraction, we conducted a frequentist network meta-analysis for each outcome at 6 h (i.e., pain relief, total pain relief [TOTPAR], summed pain intensity difference [SPID], global efficacy rating, rescue analgesia, and adverse effects). We assessed the risk of bias using a modified Cochrane RoB 2.0 tool and the certainty of evidence using the Grading of Recommendations, Assessment, Development, and Evaluation approach. We implemented the analyses in RStudio version 3.5.3 and classified interventions from most to least beneficial or harmful. We included 82 RCTs. Fifty-six RCTs enrolling 9,095 participants found moderate- and high-certainty evidence that ibuprofen 200 to 400 mg plus acetaminophen 500 to 1,000 mg (mean difference compared to placebo [MDp], 1.68; 95% confidence interval [CI], 1.06-2.31), acetaminophen 650 mg plus oxycodone 10 mg (MDp, 1.19; 95% CI, 0.85-1.54), ibuprofen 400 mg (MDp, 1.31; 95% CI, 1.17-1.45), and naproxen 400-440 mg (MDp, 1.44; 95% CI, 1.07-1.80) were most effective for pain relief on a 0 to 4 scale. Oxycodone 5 mg, codeine 60 mg, and tramadol 37.5 mg plus acetaminophen 325 mg were no better than placebo. The results for TOTPAR, SPID, global efficacy rating, and rescue analgesia were similar. Based on low- and very low-certainty evidence, most interventions were classified as no more harmful than placebo for most adverse effects. Based on moderate- and high-certainty evidence, NSAIDs with or without acetaminophen result in better pain-related outcomes than opioids with or without acetaminophen (except acetaminophen 650 mg plus oxycodone 10 mg) or placebo.

Dimerization of a specific DNA-binding protein on the DNA.

Many specific DNA-binding proteins bind to sites with dyad symmetry, and the bound form of the protein is a dimer. For some proteins, dimers form in solution and bind to DNA. LexA repressor of Escherichia coli has been used to test an alternative binding model in which two monomers bind sequentially. This model predicts that a repressor monomer should bind with high specificity to an isolated operator half-site. Monomer binding to a half-site was observed. A second monomer bound to an intact operator far more tightly than the first monomer; this cooperativity arose from protein-protein contacts.

The SOS regulatory system: control of its state by the level of RecA protease.

Our current understanding of the SOS regulatory system suggests that it can exist in two extreme states: in the repressed state, LexA protein is active, and it represses a particular set of genes called SOS genes. In the induced state, which results from various impairments to DNA replication, LexA repressor is cleaved by the specific protease activity of the RecA protein; in consequence, the SOS genes are derepressed and they express various functions that are believed to aid cell survival in induced cells. Since high levels of RecA protease activity turn on this system, it seems plausible that the level of protease activity will also control the transitions between the two states of the system. In order to assess the in vivo level of protease activity, antibody techniques were used to study the stability of LexA repressor during various phases of the SOS regulatory cycle. Repressor was reasonably stable in the repressed state, but it was degraded within a few minutes after an inducing treatment. Cleavage depended upon the RecA protease activity and resulted in the same products as seen in vitro. Cleavage preceded, and did not depend upon, derepression of any SOS gene. During the transition to the repressed state, LexA repressor became increasingly stable with time, suggesting that as DNA damage was repaired the level of protease declined. This decline depended upon derepression of the regulatory system, consistent with the belief that an inducing signal, resulting from DNA damage, reversibly activates the RecA protease and is removed by the action of one or more SOS functions. At low levels of DNA damage, a subinduced state was observed in which repressor level was reduced by a low level of cleavage. These data indicate that the level of RecA protease activity controls the state of the system and the transitions between its two states.

Highly cooperative DNA binding by the coliphage HK022 repressor.

The CI repressor protein from the temperate lambdoid phage HK022 was purified to near homogeneity and used in DNase I footprinting analyses to identify six binding sites in this phage. All these sites contained homologous 15 bp inverted repeats. Three of these 15 bp inverted repeats were located between the cI and cro (OR1 to OR3), and the other three were 3' to the cI gene (OL1 to OL3). Two of these sites were identified as operator sites for the repressor by DNA sequence analyses of virulent phage mutants. Almost all these mutations identified lay within the 15 bp inverted repeats comprising OR1 and OR2, and almost all were in the most highly conserved positions in the operators. The majority of virulent mutants contained mutations in both OR1 and OR2. Intrinsic affinities for individual operators were measured by DNase I footprinting analyses using DNA fragments which contained a single wild-type operator adjacent to two mutant operators. Comparison of these values with the affinity observed with these sites in the wild-type operator indicated that HK022 CI repressor bound cooperativity to OR1 and OR2 with a cooperativity parameter, omega, of almost 2000. Cooperative binding occurred in an alternative pairwise fashion, as previously seen with lambda CI repressor. In addition to cooperative binding between two adjacent operators, the repressor also increased the affinity for adjacent non-specific DNA sites, resulting in a periodic pattern of binding termed "phasing". This phasing pattern extended beyond regions predicted for pair-wise interaction, but was significantly decreased on a template with two adjacent operators, suggesting that pairwise cooperativity interfered with phasing.

The spacing between binding sites controls the mode of cooperative DNA-protein interactions: implications for evolution of regulatory circuitry.

The CI repressors of lambdoid phages bind cooperatively to adjacent binding sites. Although these binding sites are found at complex operators containing three binding sites, cooperative binding occurs only between dimers bound at two of the sites, a mode of binding termed pairwise cooperativity. At the level of regulation, pairwise cooperativity allows the proper operation of the genetic switch. At the mechanistic level, it has been proposed to result from steric distortion of the complex, such that a protein dimer bound to a central site cannot contact both flanking dimers because it "leans" towards one of the two sites. We have tested this model using the CI repressor of phage HK022. Previous work suggested that reducing the spacing between adjacent operators might allow cooperative interaction among three dimers, a mode of cooperativity we term extended. Using a set of synthetic templates, we have shown that reducing the spacing allows extended cooperativity among three binding sites. Accordingly, the mode of cooperativity changes qualitatively in response to changes in the inter-site spacing. That is, the change in spacing has major functional consequences for the properties of the complex. We suggest that such changes can play important roles in the evolution of gene regulatory circuitry and of other processes involving nucleoprotein complexes.

Mutations affecting cooperative DNA binding of phage HK022 CI repressor.

Cooperative protein-DNA interactions play critical roles in gene regulation in all organisms. Among the best-studied cooperative interactions is that of phage lambda repressor, which binds cooperatively to two adjacent operators. Similar cooperative interactions are also shown by several other lambdoid phage repressors, including HK022 CI repressor, which we study here. This protein has a much higher degree of cooperativity than seen with lambda repressor, and previous evidence has suggested that cooperativity may play roles in HK022 gene regulation that have no parallel in lambda. We have isolated several cooperativity or Coop- mutations in HK022 cI. These mutant proteins were partially defective in vivo for binding to two adjacent operators, but normal or nearly so for binding to a single operator. Two mutations showed mutual suppression, in that the double mutation had wild-type behavior. Analysis of several purified mutant proteins showed that they were also defective for cooperative binding in vitro. Unexpectedly, the mutant proteins showed an altered pattern of in vitro binding to DNA at non-operator sites. Several of them also increased the rate of specific repressor cleavage. We propose a conformational model in which the various functions of the wild-type protein are carried out by differing conformations; these conformations are normally in balance, and the mutations perturb this balance.

Autodigestion and RecA-dependent cleavage of Ind- mutant LexA proteins.

The LexA repressor of Escherichia coli undergoes a specific cleavage reaction in vivo, an event that leads to derepression of the SOS regulon and requires an activated form of RecA protein. In vitro, cleavage requires RecA at neutral pH; at alkaline pH, a spontaneous cleavage reaction termed autodigestion takes place. Both autodigestion and RecA-mediated cleavage cut the same bond, and are observed for the same set of substrates, suggesting that RecA acts indirectly to stimulate LexA self-cleavage at neutral pH, perhaps binding to LexA and acting as an allosteric effector. We previously isolated a set of lexA(Ind-) mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function. Here, we describe the in vitro cleavage of purified mutant proteins. All of those tested were deficient in both cleavage reactions. Although most of them were equally deficient in both reactions, some were more deficient in one reaction than the other. Several mutant proteins appeared to have defects in binding to RecA. Autodigestion of all but one of the poorly cleavable mutant proteins reached a maximum rate at pH around 10, as does wild-type LexA. The exception was KR156, which changed Lys156, a residue previously implicated in the mechanism of cleavage, to Arg, another basic residue: for this protein, the rate of autodigestion increased with pH at values above 11. RecA-mediated cleavage of KR156 was 1% the wild-type rate at pH 7, but increased with increasing pH to a plateau at pH 9.5, where the rate was 40% the wild-type rate. In contrast, an essentially constant rate was observed for wild-type LexA over the pH range 6 to 11. We suggest, first, that deprotonation of Arg156 and, by inference, Lys156 in the wild-type protein, is required for both autodigestion and RecA-mediated cleavage: and second, that RecA acts to reduce the pKa of Lys156, allowing efficient cleavage of wild-type repressor under physiological conditions.

Cooperative DNA-protein interactions. Effects of changing the spacing between adjacent binding sites.

Cooperative binding of specific DNA-binding proteins plays crucial roles in gene regulatory circuitry, and is a model system for interactions between proteins bound to DNA. We have studied coliphage HK022 repressor, which binds to two adjacent operators with a cooperativity parameter of approximately 2000. We examined the effect of changing the spacing between these two operators on cooperativity and on the conformation of the complex. Maximum cooperativity was seen with the wild-type spacing; considerable cooperativity was retained for most spacing variants, but was abolished when the operators lay on opposite faces of the DNA helix. Most spacing variants conferred changes in the conformation of the DNA-protein complex. Our data indicate that the pairwise cooperativity observed with the wild-type spacing results from a conformation that prevents protein-protein contacts with flanking bound dimers. We conclude that protein-DNA complexes involving the same specific binding sites and the same protein molecules can adopt many different conformations, depending on the spacing between the binding sites. This conclusion may be broadly applicable to protein-DNA interactions in other systems.

In vitro analysis of mutant LexA proteins with an increased rate of specific cleavage.

Specific cleavage of LexA repressor plays a crucial role in the SOS response of Escherichia coli. In vivo, cleavage requires an activated form of RecA protein. However, previous work has shown that the mechanism of cleavage is unusual, in that the chemistry of cleavage is probably carried out by residues in the repressor, and not those in RecA; RecA appears to facilitate this reaction, acting as a coprotease. We recently described a new type of lexA mutation, a class termed lexA (IndS) and here called IndS, that confers an increased rate of in vivo cleavage. Here, we have characterized the in vitro cleavage of these IndS mutant proteins, and of several double mutant proteins containing an IndS mutation and one of several mutations, termed Ind-, that decrease the rate of cleavage. We found, first, that the autodigestion reaction for the IndS mutant proteins had a higher maximum rate and a lower apparent pKa than wild-type LexA. Second, the IndS mutations had little or no effect on the rate of RecA-mediated cleavage, measured at low protein concentrations, implying that the value of Kcat/Km was unaffected. Third, the rate of autodigestion for the double-mutant proteins, relative to wild-type, was about that rate predicted from the product of the effects of the two single mutations. Finally, by contrast, these proteins displayed the same rate of RecA-mediated cleavage as did the single Ind- mutant protein. We interpret these data to mean that the IndS mutations mimic to some extent the effect of RecA on cleavage, perhaps by favoring a conformational change in LexA. We present and analyze a model that embodies these conclusions.

Effect of abx, bx and pbx mutations on expression of homeotic genes in Drosophila larvae.

We have examined the patterns of expression of the homeotic gene Ubx in imaginal discs of Drosophila larvae carrying mutations in the abx, bx and pbx regulatory domains. In haltere discs, all five bx insertion mutations examined led to a general reduction in Ubx expression in the anterior compartment; for a given allele, the strength of the adult cuticle phenotype correlated with the degree of Ubx reduction. Deletions mapping near or overlapping the sites of bx insertions, including three abx alleles and the bx34e-prv(bx-prv) allele, showed greatly reduced Ubx expression in parts of the anterior compartment of the haltere disc; however, anterior patches of strong Ubx expression often remained, in highly variable patterns. As expected, the pbx1 mutation led to reduced Ubx expression in the posterior compartment of the haltere disc; surprisingly, pbx1 also led to altered expression of the en protein near the compartment border in the central region of the disc. In the metathoracic leg, all the bx alleles caused extreme reduction in Ubx expression in the anterior regions, with no allele-specific differences. In contrast, abx and bx-prv alleles resulted in patchy anterior reductions in third leg discs. In the larval central nervous system, abx but not bx alleles affected Ubx expression; the bx-prv deletion gave a wild-type phenotype, but it could not fully complement abx mutations. In the posterior wing disc, the bx-prv allele, and to a much lesser extent the bx34e chromosome from which it arose, led to ectopic expression of Ubx. Unlike other grain-of-function mutations in the BX-C, this phenotype appeared to be partially recessive to wild type. Finally, we asked whether the ppx transformation, which results from early lack of Ubx+ function in the mesothorax and is seen in abx animals, is due to ectopic Scr expression. Some mesothoracic leg and wing discs from abx2 larvae displayed ectopic expression of Scr, which was variable in extent but always confined to the posterior compartment.

Isolation of recombinant plasmids and phage carrying the lexA gene of Escherichia coli K-12.

The lexA gene of Escherichia coli K-12 was cloned from the plasmid pLC44-14 into pBR322. Plasmids carrying lexA+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexA. The smallest lexA+ recombinant plasmid, pJL21, contained an EcoRI-PstI fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the EcoRI-PstI fragment. Plasmids homologous to pJL21, but carrying a dominant mutation, lexA3, or one of three recessive amber mutations in lexA, termed spr, were also isolated. To clone the EcoRI-PstI fragment onto a lambda vector, the PstI end was first converted to an EcoRI end by attachment of a 100-base pair PstI-EcoRI fragment isolated from the plasmid ColE1; the resultant EcoRI fragment was then cloned into the lambda vector lambda gt4. A restriction map of pLC44-14 was obtained for nine restriction enzymes. The orientation of this map was determined relative to the E. coli genetic map by complementation of the gene ubiA+ and by comparison with restriction enzyme digests of another plasmid, pLC11-9, which carries dnaB, a gene closely linked to lexA, but does not carry lexA.

Saturation mutagenesis of specific codons: elimination of molecules with stop codons from mixed pools of DNA.

In saturation mutagenesis of a protein, pools of DNA molecules are made containing a mixture of codons at a specific position. In cases where genetic methods allow screens or selections for altered function, a background of nonsense mutations can complicate genetic analysis of the resulting mutations. Methods are proposed for elimination of those molecules containing stop codons at the target codon from the pool, and for identifying positions to which these methods may be applied. Application of these methods should ensure that all changes are missense mutations, thereby simplifying genetic analysis.

Creating new restriction sites by silent changes in coding sequences.

We present methods for identifying a useful type of DNA site--one that can be mutated to create a new restriction site within a coding region without changing the amino acid sequence. These "latent sites" are abundant--silent mutations creating one of 44 different 6-bp or 8-bp recognition sites were found at relatively high density, roughly one latent site per 9 bp, in the eleven genes tested. Our analysis suggests that site-directed mutagenesis can be used to refashion coding sequences at will for flexible analysis.

Increasing CD8+ T lymphocytes predict subsequent development of intraoral lesions among individuals in the early stages of infection by the human immunodeficiency virus.

Determining the progression of human immunodeficiency virus (HIV) type 1 infection based on cellular and clinical markers has become increasingly important. Although a number of studies have shown a relationship between the presence of certain oral lesions and progression to AIDS, few data exist regarding the association with T lymphocyte counts. In this study, the question of whether intraoral lesions preceded or were the consequences of changes in T lymphocyte counts was examined. A total of 116 HIV-infected patients participating in two randomized double-blind placebo-controlled trials of zidovudine at the University of Minnesota AIDS Clinical Trials Unit (ACTU) were enrolled in a prospective dental study. Patients were examined for the presence of hairy leukoplakia, candidiasis, herpes simplex, herpes zoster, aphthae, atypical gingivitis, HIV-associated periodontitis, and necrotizing ulcerative gingivitis, as well as other oral lesions, every 3 months for a maximum of four examinations over a 1-year period. T lymphocyte counts before and after each patient's oral examination were obtained. No significant differences were found at examination 1 for differences in gender, race, age, education, tobacco smoking status, ethanol consumption habits, duration in ACTU drug protocol, duration in dental study protocol, or mean T lymphocyte counts between individuals with or without oral lesions at any time in the dental study.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease.

Specific LexA cleavage can occur under two different conditions: RecA-mediated cleavage requires an activated form of RecA, while an intramolecular self-cleavage termed autodigestion proceeds spontaneously at high pH and does not involve RecA. The two cleavage reactions are closely related. We postulate that RecA stimulates autodigestion rather than acting as a typical protease, and it is proposed to term this activity 'RecA coprotease' to emphasize this indirect role. The mechanism of autodigestion is similar to that of a serine protease, and RecA appears to act by reducing the pKa of a critical lysine residue LexA. A new class of mutants, termed lexA (IndS), is described; these mutations increase the rate of LexA cleavage.

Control of the SOS regulatory system by the level of RecA protease.

The SOS regulatory system of E. coli controls the cellular response to DNA damage and other treatments which interfere with normal DNA replication. This system can exist in two states--a repressed state, in which a set of genes (SOS genes) is repressed by the LexA repressor; and an induced state, in which the RecA protein is activated as a specific protease which cleaves LexA repressor, leading to derepression of the SOS genes. This article reviews evidence that the state of the SOS regulatory system is controlled by the level of RecA protease activity. This level is controlled in turn by a reversible activation by one or more cofactors. In vitro studies indicate that ATP or dATP and single-stranded polynucleotide are both required to activate the protease; the identity of the in vivo cofactors ("inducing signals") is not yet certain. New experiments are also described which characterize the in vivo cleavage of LexA repressor. These data support the model that the level of RecA protease controls the state of the regulatory system.

Repression of the E coli recA gene requires at least two LexA protein monomers.

To analyze the DNA binding domain of E coli LexA repressor and to test whether the repressor binds as a dimer to DNA, negative dominant lexA mutations affecting the binding domain have been isolated. A large number of amino acid substitutions between amino acid positions 39 and 46 were introduced using cassette mutagenesis. Mutants defective in DNA binding were identified and then examined for dominance to lexA+. A number of substitutions weakened repressor function partially, whereas other substitutions led to a repressor with no demonstrable activity and a defective dominant phenotype. Since the LexA binding site has dyad symmetry, we infer that this dominance results from interaction of monomers of wild-type LexA protein with mutant monomers and that an oligomeric form of repressor binds to operator. The binding of LexA protein to operator DNA was investigated further using a mutant protein, LexA408, which recognizes a symmetrically altered operator mutant but not wild-type operator. A mixture of mutant LexA408 and LexA+ proteins, but neither individual protein, bound to a hybrid recA operator consisting of mutant and wild-type operator half sites. These results suggest that at least 1 LexA protein monomer interacts with each operator half site. We discuss the role of LexA oligomer formation in binding of LexA to operator DNA.

Small airways dysfunction in influenza A virus infection: therapeutic role and potential mode of action of amantadine.

Previous studies have demonstrated alterations in gas exchange and pulmonary mechanics in presumed and documented viral upper respiratory infections. In our laboratory, using sensitive and noninvasive testing, we have demonstrated significant and prolonged alterations of pulmonary mechanics in uncomplicated natural influenza A infection, which implicate increased resistance in small airways as the cause of dysfunction. Amantadine administration in established influenza A infection has been associated with an accelerated improvement of clinical illness and pulmonary physiologic dysfunction. Preliminary study suggests that this is due to neither cholinergic blockade nor bronchodilation.

Spared descending pathways mediate locomotor recovery after subtotal spinal cord injury.

The location of pathways responsible for locomotor recovery after incomplete spinal cord injury has been studied in adult rats. Animals were allowed to recover from subtotal mid-thoracic cord section which spared left lateral and ventral funiculus fibers. Subsequent lumbar commissurotomy or left thoracic cord hemisection abolished the locomotor recovery. Spared descending fibers passing through the initial incomplete cord lesion appear to mediate locomotor recovery.

Locomotor recovery following subtotal spinal cord lesions in a rat model.

Locomotor function is described over 4 weeks in adult rats recovering from mid-thoracic spinal cord lesions, sparing the left lateral and/or ventral funiculus. A composite locomotor score was developed to follow changes by grading the swing and stance phases of locomotion, hindleg struggling withdrawal, and hopping. Motor recovery was more rapid in left than right hindlegs, consistent with sparing of left supraspinal pathways. Two temporal phases of recovery were noted in some animals, perhaps arising from two different recovery mechanisms. Motor recovery in the right hindleg was similar whether a left lateral or ventral funiculus was spared.