Hypervirulent Klebsiella pneumoniae - clinical and molecular perspectives.

Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a concerning global pathogen. hvKp is more virulent than classical K. pneumoniae (cKp) and capable of causing community-acquired infections, often in healthy individuals. hvKp is carried in the gastrointestinal tract, which contributes to its spread in the community and healthcare settings. First recognized in Asia, hvKp arose as a leading cause of pyogenic liver abscesses. In the decades since, hvKp has spread globally and causes a variety of infections. In addition to liver abscesses, hvKp is distinct from cKp in its ability to metastasize to distant sites, including most commonly the eye, lung and central nervous system (CNS). hvKp has also been implicated in primary extrahepatic infections including bacteremia, pneumonia and soft tissue infections. The genetic determinants of hypervirulence are often found on large virulence plasmids as well as chromosomal mobile genetic elements which can be used as biomarkers to distinguish hvKp from cKp clinical isolates. These distinct virulence determinants of hvKp include up to four siderophore systems for iron acquisition, increased capsule production, K1 and K2 capsule types, and the colibactin toxin. Additionally, hvKp strains demonstrate hypermucoviscosity, a phenotypic description of hvKp in laboratory conditions that has become a distinguishing feature of many hypervirulent isolates. Alarmingly, multidrug-resistant hypervirulent strains have emerged, creating a new challenge in combating this already dangerous pathogen.

Early knee changes in dancers identified by ultra-high-field 7 T MRI.

We aimed to determine whether a unique, ultra-high-field 7 T magnetic resonance imaging (MRI) scanner could detect occult cartilage and meniscal injuries in asymptomatic female dancers. This study had Institutional Review Board approval. We recruited eight pre-professional female dancers and nine non-athletic, female controls. We scanned the dominant knee on a 7 T MRI scanner using a three-dimensional fast low-angle shot sequence and a proton density, fast spin-echo sequence to evaluate cartilage and menisci, respectively. Two radiologists scored cartilage (International Cartilage Repair Society classification) and meniscal (Stoller classification) lesions. We applied two-tailed z- and t-tests to determine statistical significance. There were no cartilage lesions in dancers or controls. For the medial meniscus, the dancers demonstrated higher mean MRI score (2.38 ± 0.61 vs 1.0 ± 0.97, P < 0.0001) and higher frequency of mean grade 2 lesions (88% vs 11%, P < 0.01) compared with the controls. For the lateral meniscus, there was no difference in score (0.5 ± 0.81 vs 0.5 ± 0.78, P = 0.78) in dancers compared with the control groups. Asymptomatic dancers demonstrate occult medial meniscal lesions. Because this has been described in early osteoarthritis, close surveillance of dancers' knee symptoms and function with appropriate activity modification may help maintain their long-term knee health.

Micro-finite element analysis applied to high-resolution MRI reveals improved bone mechanical competence in the distal femur of female pre-professional dancers.

UNLABELLED: Micro-finite element analysis applied to high-resolution (0.234-mm length scale) MRI reveals greater whole and cancellous bone stiffness, but not greater cortical bone stiffness, in the distal femur of female dancers compared to controls. Greater whole bone stiffness appears to be mediated by cancellous, rather than cortical bone adaptation. INTRODUCTION: The purpose of this study was to compare bone mechanical competence (stiffness) in the distal femur of female dancers compared to healthy, relatively inactive female controls. METHODS: This study had institutional review board approval. We recruited nine female modern dancers (25.7±5.8 years, 1.63±0.06 m, 57.1±4.6 kg) and ten relatively inactive, healthy female controls matched for age, height, and weight (32.1±4.8 years, 1.6±0.04 m, 55.8±5.9 kg). We scanned the distal femur using a 7-T MRI scanner and a three-dimensional fast low-angle shot sequence (TR/TE=31 ms/5.1 ms, 0.234 mm×0.234 mm×1 mm, 80 slices). We applied micro-finite element analysis to 10-mm-thick volumes of interest at the distal femoral diaphysis, metaphysis, and epiphysis to compute stiffness and cross-sectional area of whole, cortical, and cancellous bone, as well as cortical thickness. We applied two-tailed t-tests and ANCOVA to compare groups. RESULTS: Dancers demonstrated greater whole and cancellous bone stiffness and cross-sectional area at all locations (p<0.05). Cortical bone stiffness, cross-sectional area, and thickness did not differ between groups (>0.08). At all locations, the percent of intact whole bone stiffness for cortical bone alone was lower in dancers (p<0.05). Adjustment for cancellous bone cross-sectional area eliminated significant differences in whole bone stiffness between groups (p>0.07), but adjustment for cortical bone cross-sectional area did not (p<0.03). CONCLUSIONS: Modern dancers have greater whole and cancellous bone stiffness in the distal femur compared to controls. Elevated whole bone stiffness in dancers may be mediated via cancellous, rather than cortical bone adaptation.

Psychological trauma and PTSD in HIV-positive women: a meta-analysis.

Women bear an increasing burden of the HIV epidemic and face high rates of morbidity and mortality. Trauma has been increasingly associated with the high prevalence and poor outcomes of HIV in this population. This meta-analysis estimates rates of psychological trauma and posttraumatic stress disorder (PTSD) in HIV-positive women from the United States. We reviewed 9,552 articles, of which 29 met our inclusion criteria, resulting in a sample of 5,930 individuals. The findings demonstrate highly disproportionate rates of trauma exposure and recent PTSD in HIV-positive women compared to the general population of women. For example, the estimated rate of recent PTSD among HIV-positive women is 30.0% (95% CI 18.8-42.7%), which is over five-times the rate of recent PTSD reported in a national sample of women. The estimated rate of intimate partner violence is 55.3% (95% CI 36.1-73.8%), which is more than twice the national rate. Studies of trauma-prevention and trauma-recovery interventions in this population are greatly needed.

Recent trauma is associated with antiretroviral failure and HIV transmission risk behavior among HIV-positive women and female-identified transgenders.

Trauma and posttraumatic stress disorder disproportionally affect HIV-positive women. Studies increasingly demonstrate that both conditions may predict poor HIV-related health outcomes and transmission-risk behaviors. This study analyzed data from a prevention-with-positives program to understand if socio-economic, behavioral, and health-related factors are associated with antiretroviral failure and HIV transmission-risk behaviors among 113 HIV-positive biological and transgender women. An affirmative answer to a simple screening question for recent trauma was significantly associated with both outcomes. Compared to participants without recent trauma, participants reporting recent trauma had over four-times the odds of antiretroviral failure (AOR 4.3; 95% CI 1.1-16.6; p = 0.04), and over three-times the odds of reporting sex with an HIV-negative or unknown serostatus partner (AOR 3.9; 95% CI 1.3-11.9; p = 0.02) and <100% condom use with these partners (AOR 4.5; 95% CI 1.5-13.3; p = 0.007). Screening for recent trauma in HIV-positive biological and transgender women identifies patients at high risk for poor health outcomes and HIV transmission-risk behavior.

A bacterial enhancer functions to tether a transcriptional activator near a promoter.

The nitrogen regulatory protein NtrC of enteric bacteria activates transcription of the glnA gene by catalyzing isomerization of closed complexes between RNA polymerase and the glnA promoter to open complexes. NtrC binds to sites upstream of glnA that have properties of eukaryotic transcriptional enhancers. NtrC-binding sites were found to facilitate open complex formation when these sites and the glnA promoter were located on different rings of a singly linked catenane, but not when the two rings were decatenated. The results provide evidence that NtrC contacts RNA polymerase-promoter complexes in a process mediated by formation of a DNA loop. NtrC-binding sites serve to tether NtrC near the glnA promoter, thereby increasing the frequency of collisions between NtrC and polymerase-promoter complexes.

Prokaryotic transcriptional enhancers and enhancer-binding proteins.

A number of prokaryotic enhancer-binding proteins activate transcription by specialized forms of RNA polymerase. The enhancer-binding proteins catalyse isomerization of the initial complex formed between RNA polymerase and a promoter from the closed to the open state. To do so, one class of enhancer-binding proteins contacts its cognate polymerase by DNA loop formation but the other, which is represented by a single member, does not. Despite this difference, both classes of enhancer-binding proteins must hydrolyse ATP to catalyse open complex formation.

alpha1beta2delta, a silent GABAA receptor: recruitment by tracazolate and neurosteroids.

BACKGROUND AND PURPOSE: This study investigated the alpha(1)beta(2)delta isoform of the GABA(A) receptor that is presumably expressed in the forebrain. The functional and pharmacological properties of this receptor combination are largely unknown. EXPERIMENTAL APPROACH: We expressed alpha(1)beta(2)delta GABA(A) receptors in Xenopus laevis oocytes. GABA-activated currents, in the presence and absence of modulators, were recorded using the two-electrode voltage clamp technique. KEY RESULTS: The alpha(1)beta(2)delta isoform of the GABA(A) receptor exhibited an extremely small GABA-mediated current. Tracazolate increased the current amplitude evoked by a half-maximal concentration (EC(50)) of GABA by 59-fold. The maximum current was increased 23-fold in the presence of a saturating GABA concentration. Concomitant with the increase in the maximum, was a 4-fold decrease in the EC(50). Finally, a mutation in the second transmembrane domain of the delta subunit that increases receptor efficacy (L286S), eliminated the increase in the maximum GABA-activated current. The endogenous neurosteroid, tetrahydrodeoxycorticosterone (THDOC), also decreased the EC(50) and increased the maximum current amplitude, although to a lesser degree than that of tracazolate. CONCLUSIONS AND IMPLICATIONS: Taken all together, these findings indicate that the small GABA-mediated currents in the absence of the modulator are due to a low efficacy for activation. In the absence of modulators, alpha(1)beta(2)delta GABA receptors would be effectively silent and therefore contribute little to inhibition in the CNS. In the presence of tracazolate or endogenous neurosteroids however, this particular receptor isoform could exert a profound inhibitory influence on neuronal activity.

Channel opening locks agonist onto the GABAC receptor.

Determination of the activation mechanism of neurotransmitter-operated ion channels has been hindered by a limited understanding of the relationship between agonist binding and the gating of the integral ion pore. Here we describe a [3H]ligand binding assay that enables us to make repeated binding measurements from the same intact oocyte expressing recombinant human rho 1 GABAC receptors and directly correlate the binding kinetics with electrophysiological measurements. We have determined an association rate for GABA of about 10(5) M-1s-1; this is four orders of magnitude slower than diffusion, indicating GABA has restricted access to its binding site. We also demonstrate that GABA dissociates at two rates. Our data are consistent with the faster rate being the true microscopic dissociation rate of GABA, with the slower rate occurring because the opening of the pore detains agonist release.

Glutamate at the site of phosphorylation of nitrogen-regulatory protein NTRC mimics aspartyl-phosphate and activates the protein.

The NTRC protein of enteric bacteria is an enhancer-binding protein that activates transcription by the sigma 54-holoenzyme form of RNA polymerase under nitrogen-limiting conditions. In vitro NTRC must be phosphorylated to catalyze ATP hydrolysis and activate transcription. The site of phosphorylation of NTRC from Salmonella typhimurium is Aspartate 54, which lies in the amino-terminal regulatory domain of the protein. We used site-directed mutagenesis to make "conservative" substitutions at residue 54 to alanine, asparagine, and glutamate, and examined the properties of the mutant NTRC proteins in vitro and in vivo. In vitro none of them was detectably phosphorylated, as expected if D54 is, in fact, the sole site of phosphorylation. D54A and D54N did not activate transcription of glnA but, interestingly, D54E activated constitutively. Activation by D54E was partial compared to that by phosphorylated wild-type NTRC. Combining D54A or D54N with S160F, a change in the central domain of NTRC that partially bypasses the requirement for phosphorylation, yielded doubly mutant proteins that were as active as a form carrying S160F alone, indicating that the changes in D54 did not adversely affect the function of the remainder of NTRC. Combining D54E with S160F increased the levels of constitutive ATPase activity and transcriptional activation above those of mutant NTRC proteins carrying either single change alone. We conclude that phosphorylation of aspartate 54 is required to activate NTRC and postulate that the D54E mutation mimics phosphorylation, thereby allowing NTRC to hydrolyze ATP and activate transcription. Phenotypes of mutant strains encoding NTRC proteins with substitutions at D54 indicated that phosphorylation of NTRC at position 54 was necessary for normal growth in the absence of glutamine and that such phosphorylation occurred to some extent even in the absence of NTRB.

Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in the central activation domain.

Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding protein that activates transcription by the sigma 54-holoenzyme. To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer. In this paper we characterize "constitutive" mutant forms of the NtrC protein from Salmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated. The amino acids altered in NtrCconstitutive proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation. The residues that are altered are not conserved among activators of the sigma 54-holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria). NtrCconstitutive proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription. Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms. Removal of the N-terminal regulatory domain from two NtrCconstitutive proteins with amino acid substitutions in the central domain (NtrCS160F and NtrCV2881) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain. This conclusion is confirmed by the observation that the ATPase activity of delta N-NtrCS160F is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding. Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain.

Repressor forms of the enhancer-binding protein NrtC: some fail in coupling ATP hydrolysis to open complex formation by sigma 54-holoenzyme.

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein that activates transcription by catalyzing isomerization of closed complexes between sigma54-holoenzyme and a promoter to open complexes. To catalyze this reaction, NtrC must be phosphorylated and form an appropriate oligomer so that it can hydrolyze ATP. NtrC can also repress transcription by sigma70-holoenzyme. In this paper we characterize "repressor" mutant forms of NtrC from Salmonella typhimurium, forms that have lost the ability to activate transcription by sigma54-holoenzyme (in vitro activity at least 1000-fold lower than wild-type) but retain the ability to repress transcription by sigma70-holoenzyme. The amino acid substitutions in NtrCrepressor proteins that were obtained by classical genetic techniques alter residues in the central domain of the protein, the domain directly responsible for transcriptional activation. Commensurate with this, phosphorylation and the autophosphatase activities of NtrCrepressor proteins, which are functions of the amino-terminal regulatory domain of NtrC, are normal. In addition, these proteins have essentially normal DNA-binding, which is a function of the C-terminal region of NtrC and bind cooperatively to enhancers. (The NtrC(G219K) protein has "improved" DNA-binding, which is discussed.) We previously presented evidence that several NtrCrepressor proteins have impaired ATPase activity. We now show that two other repressor proteins, NtrC(A216V) and NtrC(A220T), have as much ATPase activity as wild-type NtrC when they are phosphorylated and bound to an enhancer and that they have considerably more activity than an unphosphorylated NtrC(constitutive) protein, which is capable of activating transcription. These results demonstrate that NtrC(A216V) and NtrC(A220T) fail in a function of the central domain other than ATPase activity. Although they may fail in contact with sigma54-holoenzyme per se, the fact that alanine is the amino acid normally found at these positions leads us to speculate that these proteins fail in coupling energy to a change in conformation of the polymerase.

Brief psychotherapy of bereavement reactions. The relationship of process to outcome.

We studied the relationship of dispositional and process variables with outcome in 52 bereaved patients given time-limited dynamic psychotherapy. Outcomes were generally favorable in symptom relief and improvement in relationship and occupational functioning. Patients' symptoms improved more than did their social and work functioning. Pretreatment levels of impairment or distress were significantly related to outcome, but most demographic and dispositional variables did not predict outcome. Process variables examined in relation to outcome--therapeutic alliance and actions by the therapist--were not significantly related to either type of outcome. When we considered the same process variables in interaction with two dispositional variables, motivation for dynamic therapy and developmental level of the self-concept, we found significant predictions of outcome. The major findings suggest that more exploratory actions were more suitable for highly motivated and/or better-organized patients and less suitable for patients with lower levels of motivation or organization of self-concept. More supportive actions were more suitable for patients at lower dispositional levels and less therapeutic for patients at higher levels.

Patterns of individual change scales.

Measurement of complex modifications in personal functioning is a necessary step to the relevant translation of psychotherapy research into clinical practice. Using an assessment strategy aimed toward decreasing subjective bias, we pursued an inductive clinical approach in constructing the Patterns of Individual Change Scales (PICS) battery. Designed for use with a bereaved population, each of the 13 scales has a unitary focus and hierarchy of functional levels across the domains of symptoms, social relations, and self-concept. Nine experienced clinical judges rated the scales after viewing videotaped evaluation interviews of 18 patients and 18 field subject. In general, the PICS had a suitable range for our borderline through normal population, reached acceptable levels of reliability, and could discriminate among subjects in clinically meaningful ways. Future analyses will consider issues of bias, validity, and generalization.

A proposed method for measuring change beyond symptoms.

The initial report of the Patterns of Individual Change Scales battery presented the rationale, development, and conceptualization of this measurement package. Empirical data supported the reliability and validity of the scales as measures of patient characteristics that extended beyond the symptomatic domain. This article presents more extensive reliability and validity data. Ratings on two new samples were collected, and the interrater reliability coefficients for the scales were again of appreciable magnitude. Discriminant validity was examined, as was the factor structure of the battery. The results suggested that the scales tap three distinct sources of variance. Comparisons with symptom measures and case illustrations indicated that the measurement of changes in patients' functioning beyond symptoms is necessary and viable.

Judging change in psychotherapy. Reliability of clinical formulations.

Careful clinical formulation describing the dimensions of core neurotic conflicts is integral to the practice of psychodynamic psychotherapy. Therefore, a method for reliable and valid formulation is a necessary precursor for the empirical investigation of the impact of such therapy. Our study evaluated a method of outcome assessment based on clinical formulations. Results from 18 patients indicate that independently derived formulations of a central conflict do not agree and that these disagreements in formulation are reflected in disagreements in the change ratings that are based on them. Additional analyses suggested that symptomatic change represents the major component in outcome rating.

Allosteric activation mechanism of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine.

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha 1 beta 2 gamma 2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma 2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta 2Y157S; increased the EC(50)) and the conserved M2 leucine (beta 2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.

Toll-like receptor-2 transduces signals for NF-kappa B activation, apoptosis and reactive oxygen species production.

The innate immune system coordinates the inflammatory response to pathogens. To do so, cells of the innate immune system must rapidly discriminate between self and non-self. All bacteria express membrane-associated lipoproteins. These molecules activate cells of the innate immune system to initiate host defense mechanisms. However, it is currently unknown how the innate immune system recognizes bacterial lipoproteins. Here, we describe that in response to bacterial lipoprotein, human Toll-like receptor-2 activates three different cellular responses: nuclear factor-kappaB dependent transcription, programmed cell death and reactive oxygen species production. We propose that Toll-like receptor-2 fulfils multiple roles in the genesis of the immune response to bacterial pathogens.

Sensory gating in chronic posttraumatic stress disorder: reduced auditory P50 suppression in combat veterans.

BACKGROUND: Posttraumatic stress disorder (PTSD) may be associated with a general impairment of cognitive function that extends beyond the processing of trauma-specific stimuli. Suppression of the auditory P50 response to repeated stimuli occurs in normal subjects and reflects the central nervous system's ability to screen out repetitive stimuli, a phenomenon referred to as sensory gating. This study examines P50 sensory gating to nonstartle auditory stimuli in PTSD subjects and normal controls. METHODS: P50 generation and gating were studied using a conditioning/testing paradigm in 15 male subjects with PTSD and 12 male controls. P50 test/conditioning (T/C) ratios were estimated using the Singular Value Decomposition method. RESULTS: The amplitude of the P50 response to the conditioning stimulus did not differ in subjects with PTSD compared to normal controls. The P50 T/C ratio is increased in PTSD subjects (mean = .408, SD = .275) as compared to the controls (mean = .213, SD = .126, two tailed t, p = .024). CONCLUSIONS: This study provides evidence that PTSD is associated with impaired gating to nonstartle trauma-neutral auditory stimuli.

Decreased hippocampal N-acetylaspartate in the absence of atrophy in posttraumatic stress disorder.

BACKGROUND: Previous magnetic resonance imaging studies of posttraumatic stress disorder reported hippocampal volume loss. The goals of this study were 1) to determine the relationship between hippocampal atrophy and posttraumatic stress disorder in the absence of alcohol abuse, and 2) to test if loss of N-acetylaspartate (a neuron marker) in the hippocampus of posttraumatic stress disorder occurs separate from atrophy. In addition, volume changes in the entorhinal cortex were also explored. METHODS: Eighteen male patients with combat-related posttraumatic stress disorder (mean age 51.2 +/- 2.5 years) and 19 male control subjects (mean age 51.8 +/- 3.2 years) were studied using magnetic resonance imaging and Proton magnetic resonance spectroscopic imaging. Both groups had no alcohol and drug abuse during the past 5 years. RESULTS: Posttraumatic stress disorder and control subjects had similar volumes of hippocampus and entorhinal cortex. In contrast to volume, N-acetylaspartate was significantly reduced by about 23% bilaterally in the hippocampus of posttraumatic stress disorder when compared with control subjects, and creatine-containing compounds were reduced by 26% in the right hippocampus of posttraumatic stress disorder. CONCLUSIONS: N-acetyl asparate and creatine reductions imply that there are hippocampal abnormalities in posttraumatic stress disorder. Furthermore, these metabolite changes seem to be better indicators of posttraumatic stress disorder pathology than volume losses.