Rapid Immunofluorescence Microscopy for Diagnosis of Melioidosis

An immunofluorescent (IF) method that detects Burkholderia pseudomallei in clinical specimens within 10 min was devised. The results of this rapid method and those of an existing IF method were prospectively compared with the culture results for 776 specimens from patients with suspected melioidosis. The sensitivities of both IF tests were 66%, and the specificities were 99.5 and 99.4%, respectively.     

复合树脂预热的研究现状和临床应用

复合树脂作为临床常用牙科充填材料存在耐磨性差、聚合收缩大和牙本质边缘适应性不佳等问题。近年有研究发现预热可以改善复合树脂的性能,本文就预热复合树脂对其单体转化率、边缘微渗漏、机械性能以及牙髓的影响等方面进行探讨,以供临床参考。     

Recurrent melioidosis in patients in northeast Thailand is frequently due to reinfection rather than relapse

Human melioidosis is associated with a high rate of recurrent disease, despite adequate antimicrobial treatment. Here, we define the rate of relapse versus the rate of reinfection in 116 patients with 123 episodes of recurrent melioidosis who were treated at Sappasithiprasong Hospital in Northeast Thailand between 1986 and 2005. Pulsed-field gel electrophoresis was performed on all isolates; isolates from primary and recurrent disease for a given patient different by one or more bands were examined by a sequence-based approach based on multilocus sequence typing. Overall, 92 episodes (75%) of recurrent disease were caused by the same strain (relapse) and 31 episodes (25%) were due to infection with a new strain (reinfection). The interval to recurrence differed between patients with relapse and reinfection; those with relapses had a median time to relapse of 228 days (range, 15 to 3,757 days; interquartile range [IQR], 99.5 to 608 days), while those with reinfection had a median time to reinfection of 823 days (range, 17 to 2,931 days; IQR, 453 to 1,211 days) (P = 0.0001). A total of 64 episodes (52%) occurred within 12 months of the primary infection. Relapse was responsible for 57 of 64 (89%) episodes of recurrent infection within the first year after primary disease, whereas relapse was responsible for 35 of 59 (59%) episodes after 1 year (P < 0.0001). Our data indicate that in this setting of endemicity, reinfection is responsible for one-quarter of recurrent cases. This finding has important implications for the clinical management of melioidosis patients and for antibiotic treatment studies that use recurrent disease as a marker for treatment failure.     

Ultrasound-Guided Versus Landmark-Guided Local Corticosteroid Injection for Carpal Tunnel Syndrome: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Objective

To review the literature and assess the comparative effectiveness of ultrasound-guided versus landmark-guided local corticosteroid injections in patients with carpal tunnel syndrome (CTS).

Surveillance of Artemisinin Resistance in Plasmodium falciparum in India Using the kelch13 Molecular Marker

Malaria treatment in Southeast Asia is threatened with the emergence of artemisinin-resistant Plasmodium falciparum. Genome association studies have strongly linked a locus on P. falciparum chromosome 13 to artemisinin resistance, and recently, mutations in the kelch13 propeller region (Pfk-13) were strongly linked to resistance. To date, this information has not been shown in Indian samples. Pfk-13 mutations were assessed in samples from efficacy studies of artemisinin combination treatments in India. Samples were PCR amplified and sequenced from codon 427 to 727. Out of 384 samples, nonsynonymous mutations in the propeller region were found in four patients from the northeastern states, but their presence did not correlate with ACT treatment failures. This is the first report of Pfk-13 point mutations from India. Further phenotyping and genotyping studies are required to assess the status of artemisinin resistance in this region.     

From the Cover: Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.Hyperpolarization-activated cyclic nucleotide-gated (HCN1–4) channels are the molecular determinants of the h-current (I_h), which regulates critical neuronal properties, including membrane resting potential, dendritic excitability, and intrinsic rhythmicity (1). HCN channels are dually regulated by voltage and by binding of cAMP to the cyclic nucleotide binding domain (CNBD), which is found on the cytoplasmic C-terminal tail of the channel. The CNBD exerts a tonic inhibition on the channel pore, with the opening transition of the channel being allosterically coupled to the conformational changes in the CNBD induced by cAMP binding (2). Thus, the closed-to-open transition of the channel is thought to reflect the transition from the cAMP-free conformation to the cAMP-bound conformation of the CNBD, which stabilize, respectively, the closed and open states of the channel (2, 3). The C-linker, an α-helical folded domain that connects the CNBD to the pore region, conveys the regulation of channel gating from the CNBD to the pore (4–6). As a result of this allosteric mechanism, the binding of cAMP shifts the voltage dependence of the HCN channel opening to more positive potentials and increases maximal I_h at extreme negative voltages, where voltage gating is complete.In addition to cAMP, HCN channels in the brain are regulated by auxiliary proteins, such as TRIP8b, a cytosolic β-subunit of neuronal HCN channels, which inhibits channel activation by antagonizing the effects of cAMP (7–9). We have previously shown that TRIP8b_core, an 80-aa sequence located in the TRIP8b protein core that directly interacts with the C-linker/CNBD region of HCN channels, is necessary and sufficient to prevent all of the effects of cAMP on the channel (10, 11). TRIP8b_core decreases both the sensitivity of the channel to cAMP [half maximal concentration (k_1/2)] and the efficacy of cAMP in inducing channel opening [half activation voltage (V_1/2)]; conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic effects are well described by a cyclic allosteric model in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state (11).A second important action of TRIP8b is to reduce maximal current through HCN channels in the absence of cAMP (11). As a consequence, application of cAMP produces a larger increase in maximal I_h in the presence of TRIP8b than in its absence. The observation that TRIP8b exerts opposing influences on the two major actions of cAMP on HCN channel function, namely, reduces the effect of cAMP to shift the voltage dependence of channel gating but enhances the effect of cAMP to increase maximal current, has important implications for the ability of cAMP to modulate neuronal excitability in vivo. Thus, the relative extent by which neuromodulatory transmitters alter maximal I_h or shift the voltage dependence of HCN channel gating can vary widely among distinct classes of neurons (12–14). The differential expression of TRIP8b may provide a mechanistic explanation for this finding, because in neurons with high levels of TRIP8b expression, cAMP will exert a larger action to enhance maximal current, and a smaller action to alter the voltage dependence of channel gating, compared with neurons in which TRIP8b expression is low. Such fine-tuning broadens the range of physiological actions that cAMP can exert to modulate neuronal firing.In the present study, we address the structural basis for the mutually antagonistic effects of cAMP and TRIP8b on HCN channel function. Although our previous biochemical and electrophysiological data strongly support the hypothesis that TRIP8b and cAMP binding sites do not overlap, direct structural information on the TRIP8b–CNBD complex is required to validate the allosteric antagonism model of interaction between the two ligands. A plausible hypothesis for the antagonistic effect of TRIP8b and cAMP is that each of the two ligands stabilizes the CNBD in a conformation that decreases the affinity for the other. To test this hypothesis, we first generated the 3D structure of the cAMP-free HCN2 channel CNBD using solution NMR spectroscopy and then characterized its interaction with the TRIP8b_core fragment. By comparing the cAMP-free with the available cAMP-bound HCN2 channel CNBD structure (15, 16), we reconstruct the full conformational changes induced by cAMP binding, revealing critical transitions occurring in the P- and C-helices of the CNBD, and further highlighting the role of the N-terminal helical bundle in transducing the movements of the CNBD to the channel pore. We next identify, through NMR titration, site-directed mutagenesis, and biochemical interaction assays, the binding site of TRIP8b_core on the cAMP-free form of the HCN2 channel CNBD. Our results demonstrate that cAMP and TRIP8b do not directly compete for the same binding region and support a model of mutual allosteric inhibition between cAMP and TRIP8b. Finally, our results clarify the mechanism by which TRIP8b antagonizes the effect of cAMP on channel gating: TRIP8b directly interacts with two mobile elements that drive the ligand-induced conformational changes in the CNBD. TRIP8b binding to the CNBD therefore prevents the cAMP-induced transition and stabilizes the channel in the cAMP-free conformation.     

FGF21 is increased by inflammatory stimuli and protects leptin-deficient ob/ob mice from the toxicity of sepsis

The acute phase response (APR) produces marked alterations in lipid and carbohydrate metabolism including decreasing plasma ketone levels. Fibroblast growth factor 21 (FGF21) is a recently discovered hormone that regulates lipid and glucose metabolism and stimulates ketogenesis. Here we demonstrate that lipopolysaccharide (LPS), zymosan, and turpentine, which induce the APR, increase serum FGF21 levels 2-fold. Although LPS, zymosan, and turpentine decrease the hepatic expression of FGF21, they increase FGF21 expression in adipose tissue and muscle, suggesting that extrahepatic tissues account for the increase in serum FGF21. After LPS administration, the characteristic decrease in plasma ketone levels is accentuated in FGF21-/- mice, but this is not due to differences in expression of carnitine palmitoyltransferase 1α or hydroxymethyglutaryl-CoA synthase 2 in liver, because LPS induces similar decreases in the expression of these genes in FGF21-/- and control mice. However, in FGF21-/- mice, the ability of LPS to increase plasma free fatty acid levels is blunted. This failure to increase plasma free fatty acid could contribute to the accentuated decrease in plasma ketone levels because the transport of fatty acids from adipose tissue to liver provides the substrate for ketogenesis. Treatment with exogenous FGF21 reduced the number of animals that die and the rapidity of death after LPS administration in leptin-deficient ob/ob mice and to a lesser extent in control mice. FGF21 also protected from the toxic effects of cecal ligation and puncture-induced sepsis. Thus, FGF21 is a positive APR protein that protects animals from the toxic effects of LPS and sepsis.     

Immunization with heat-killed Francisella tularensis LVS elicits protective antibody-mediated immunity

Francisella tularensis (FT) has been classified by the CDC as a category A pathogen because of its high virulence and the high mortality rate associated with infection via the aerosol route. Because there is no licensed vaccine available for FT, development of prophylactic and therapeutic regimens for the prevention/treatment of infection is a high priority. In this report, heat-killed FT live vaccine strain (HKLVS) was employed as a vaccine immunogen, either alone or in combination with an adjuvant, and was found to elicit protective immunity against high-dose FT live vaccine strain (FTLVS) challenge. FT-specific antibodies produced in response to immunization with HKLVS alone were subsequently found to completely protect naive mice against high-dose FT challenge in both infection-interference and passive immunization experiments. Additional passive immunization trials employing serum collected from mice immunized with a heat-killed preparation of an O-antigen-deficient transposon mutant of FTLVS (HKLVS-OAg(neg)) yielded similar results. These findings demonstrated that FT-specific antibodies alone can confer immunity against high-dose FTLVS challenge, and they reveal that antibody-mediated protection is not dependent upon production of LPS-specific antibodies.     

The Building Blocks Collaborative: Advancing a Life Course Approach to Health Equity Through Multi-Sector Collaboration

Too many children are born into poverty, often living in disinvested communities without adequate opportunities to be healthy and thrive. Two complementary frameworks—health equity and life course—propose new approaches to these challenges. Health equity strategies seek to improve community conditions that influence health. The life course perspective focuses on key developmental periods that can shift a person’s trajectory over the life course, and highlights the importance of ensuring that children have supports in place that set them up for long-term success and health. Applying these frameworks, the Alameda County Public Health Department launched the Building Blocks Collaborative (BBC), a countywide multi-sector initiative to engage community partners in improving neighborhood conditions in low-income communities, with a focus on young children. A broad cross-section of stakeholders, called to action by the state of racial and economic inequities in children’s health, came together to launch the BBC and develop a Bill of Rights that highlights the diverse factors that contribute to children’s health. BBC partners then began working together to improve community conditions by learning and sharing ideas and strategies, and incubating new collaborative projects. Supportive health department leadership; dedicated staff; shared vision and ownership; a flexible partnership structure; and broad collective goals that build on partners’ strengths and priorities have been critical to the growth of the BBC. Next steps include institutionalizing BBC projects into existing infrastructure, ongoing partner engagement, and continued project innovation—to achieve a common vision that all babies have the best start in life.