Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution

Thiamine pyrophosphate (TPP)-sensitive mRNA domains are the most prevalent riboswitches known. Despite intensive investigation, the complex ligand recognition and concomitant folding processes in the TPP riboswitch that culminate in the regulation of gene expression remain elusive. Here, we used single-molecule fluorescence resonance energy transfer imaging to probe the folding landscape of the TPP aptamer domain in the absence and presence of magnesium and TPP. To do so, distinct labeling patterns were used to sense the dynamics of the switch helix (P1) and the two sensor arms (P2/P3 and P4/P5) of the aptamer domain. The latter structural elements make interdomain tertiary contacts (L5/P3) that span a region immediately adjacent to the ligand-binding site. In each instance, conformational dynamics of the TPP riboswitch were influenced by ligand binding. The P1 switch helix, formed by the 5′ and 3′ ends of the aptamer domain, adopts a predominantly folded structure in the presence of Mg²⁺ alone. However, even at saturating concentrations of Mg²⁺ and TPP, the P1 helix, as well as distal regions surrounding the TPP-binding site, exhibit an unexpected degree of residual dynamics and disperse kinetic behaviors. Such plasticity results in a persistent exchange of the P3/P5 forearms between open and closed configurations that is likely to facilitate entry and exit of the TPP ligand. Correspondingly, we posit that such features of the TPP aptamer domain contribute directly to the mechanism of riboswitch-mediated translational regulation.     

Neonatal hyperthyrotropinemia: population characteristics,diagnosis, management and outcome after cessation of therapy

Introduction Neonatal hyperthyrotropinaemia (HT), defined by elevated TSH and normal T₄, is either transient or persistent. The eventual outcome of neonatal HT is unpredictable and the management of HT patients is controversial. We assessed perinatal parameters and diagnostic measures that may distinguish between transient and persistent HT, compared with congenital hypothyroidism (CH). We also aimed to recommend optimal treatment in these forms of thyroid impairment. Design and patients A multi‐centre, retrospective study was conducted in six paediatric endocrinology units. Forty‐three HT patients and 83 CH patients were included in the study. Measurements We evaluated differences in birth weight (BW), gestational age (GA), modes of diagnosis, screening and confirmatory T₄ and TSH levels, thyroid imaging results and optimal thyroxine doses between HT and CH and between the two forms of HT. Results Newborns with HT had lower BW and GA than those with CH. Transient (n = 18) and persistent HT (n = 25) patients were indistinguishable by most parameters, but those with persistent HT had a higher prevalence of abnormal thyroid imaging (69%vs 8%; P = 0·005). During treatment, 79% and 55% of transient and persistent HT patients respectively experienced elevated levels of free T_4. Although most HT patients were reevaluated after 2·5 years, six transient HT patients stopped therapy and showed full recovery within the first year of life. Conclusions We recommend obtaining thyroid imaging to distinguish between the two forms of HT. Adherence to recommended doses of thyroxine and probably early cessation of therapy in transient HT can prevent iatrogenic hyperthyroidism in these patients.     

Using a telephone support group for HIV-positive persons aged 50+ to increase social support and health-related knowledge

Middle-aged and older persons living with HIV/AIDS have unique needs arising from the physical, mental, and social changes associated not only with normal aging but also related to living with a chronic illness. To address these needs, two 10-week telephone psychoeducational support groups were offered for HIV-infected persons aged 50 or older. Each group was cofacilitated by a registered nurse and a social worker; each session was 50-60 minutes every Friday; approximately 1-5 clients participated with an average number of 3 clients and there was no charge to the participants. The issues addressed in the group were: (1) staying healthy; (2) symptom management; (3) understanding other chronic illnesses; (4) understanding diagnostic tests; (5) strategies for effective interactions with the health care provider; (6) optimizing HIV/AIDS medication use; (7) understanding new developments in HIV treatment; (8) coping with losses; and (9) finding commonalities. There were unique challenges. Boundaries of respect were more difficult to maintain in a teleconference, as opposed to an in-person group. Nonverbal cues were impossible to interpret and therefore greater sensitivity was required to gauge the impact of borderline, less controlled group members, especially in relationship to other group members who may tend to be less assertive. One group member withdrew because his hearing was impaired and the telephone modality was just too challenging. It has been found that middle-aged and older adults living with HIV/AIDS with greater depression identify a need for information and support. It is crucial to share concrete information, identify symptoms clearly, and explore the use of effective and ineffective medications and treatments. The psychosocial concerns are very real and encouraging group members to open up to one another to create a cohesive community of sharing is equally important. Although the use of teleconference technology makes this more difficult, the attempt to create a situation that facilitates connections with another individual is one strategy to decrease geographic or logistical isolation.     

Glibenclamide treatment in permanent neonatal diabetes mellitus due to an activating mutation in Kir6.2

Permanent neonatal diabetes mellitus (PNDM) is a rare form of diabetes characterized by insulin-requiring hyperglycemia that is diagnosed within the first months of life. Recently, activating mutations in the gene encoding the ATP-sensitive potassium-channel subunit Kir6.2 were identified in 10 PNDM patients. Tolbutamide-stimulated insulin secretion, demonstrated in 3 of these patients suggested that some PNDM patients may respond to oral sulfonylurea treatment. In this report, we describe an infant with PNDM due to an arginine-to-histidine substitution at position 201 (R201H) of the gene encoding Kir6.2. After insulin pump therapy for six months, he was shifted to oral glybenclamide therapy at a daily dose of 0.8 mg/kg. Basal c-peptide level increased by two fold during glybenclamide treatment, but no further elevation was observed following intravenous glucose administration. Outpatient, continuous glucose monitoring while on a normal infant diet demonstrated a marked improvement in glycemic control. This study demonstrates the feasibility of oral sulfonylurea treatment in PNDM patients with Kir6.2 mutations even during infancy, and the superiority of this approach over insulin administration.     

Injurious effect of the eosinophil peroxide-hydrogen peroxide-halide system and major basic protein on human nasal epithelium in vitro

Tissue injury is observed in allergic and nonallergic eosinophilic rhinitis, but the mechanism of this injury is unclear. Because eosinophils are prominent in biopsy specimens in these conditions, we hypothesized that they may participate in the injury process. Initially, we developed techniques to isolate and purify human nasal epithelial cells from turbinate biopsies to use as target cells for eosinophil granule products. Primary cultures from explants were characterized by electron microscopy and indirect immunofluorescence with a panel of primary monoclonal and polyclonal antibodies. These studies revealed the homogeneity of the cells and confirmed their epithelial nature. Cultured nasal epithelial cells were then exposed to either purified human eosinophil peroxidase, bromide, and glucose plus glucose oxidase, as a continuous source of hydrogen peroxide, or eosinophil major basic protein. Neither eosinophil peroxidase alone nor glucose plus glucose oxidase in the absence of eosinophil peroxidase were injurious, but the combined addition of eosinophil peroxidase, glucose/glucose oxidase, and bromide produced marked target cell lysis. This effect was time- and eosinophil peroxidase dose-dependent. Catalase and azide significantly inhibited the lysis of these cells, suggesting the eosinophil peroxidase-catalyzed products of halide oxidation mediated this form of injury. The addition of purified human eosinophil major basic protein also caused dose- and time-dependent lysis of the nasal epithelial cells but required longer incubation periods to effect injury. We hypothesize that the eosinophil peroxidase-hydrogen peroxide-halide system and major basic protein may injure the nasal epithelium in inflammatory conditions such as allergic and nonallergic eosinophilic rhinitis.