Role of helical constraints of the EBS1-IBS1 duplex of a group II intron on demarcation of the 5' splice site.

Recognition of the 5' splice site by group II introns involves pairing between an exon binding sequence (EBS) 1 within the ID3 stem-loop of domain 1 and a complementary sequence at the 3' end of exon 1 (IBS1). To identify the molecular basis for splice site definition of a group IIB ai5γ intron, we probed the solution structure of the ID3 stem-loop alone and upon binding of its IBS1 target by solution NMR. The ID3 stem was structured. The base of the ID3 loop was stacked but displayed a highly flexible EBS1 region. The flexibility of EBS1 appears to be a general feature of the ai5γ and the smaller Oceanobacillus iheyensis (O.i.) intron and may help in effective search of conformational space and prevent errors in splicing as a result of fortuitous base-pairing. Binding of IBS1 results in formation of a structured seven base pair duplex that terminates at the 5' splice site in spite of the potential for additional A-U and G•U pairs. Comparison of these data with conformational features of EBS1-IBS1 duplexes extracted from published structures suggests that termination of the duplex and definition of the splice site are governed by constraints of the helical geometry within the ID3 loop. This feature and flexibility of the uncomplexed ID3 loop appear to be common for both the ai5γ and O.i. introns and may help to fine-tune elements of recognition in group II introns.

NMR structure of the 5' splice site in the group IIB intron Sc.ai5γ--conformational requirements for exon-intron recognition.

A crucial step of the self-splicing reaction of group II intron ribozymes is the recognition of the 5' exon by the intron. This recognition is achieved by two regions in domain 1 of the intron, the exon-binding sites EBS1 and EBS2 forming base pairs with the intron-binding sites IBS1 and IBS2 located at the end of the 5' exon. The complementarity of the EBS1•IBS1 contact is most important for ensuring site-specific cleavage of the phosphodiester bond between the 5' exon and the intron. Here, we present the NMR solution structures of the d3' hairpin including EBS1 free in solution and bound to the IBS1 7-mer. In the unbound state, EBS1 is part of a flexible 11-nucleotide (nt) loop. Binding of IBS1 restructures and freezes the entire loop region. Mg(2+) ions are bound near the termini of the EBS1•IBS1 helix, stabilizing the interaction. Formation of the 7-bp EBS1•IBS1 helix within a loop of only 11 nt forces the loop backbone to form a sharp turn opposite of the splice site, thereby presenting the scissile phosphate in a position that is structurally unique.

Vitamin D Deficiency and Unclear Abdominal Pain in Patients from Low- and Middle-Income Countries.

Background: Abdominal pain is one of the commonest symptoms in emergency departments (EDs). Diagnosis demands full attention and critical thinking, since many diseases manifest atypically and the consequences of overlooking the symptoms may be disastrous. Despite intensive diagnostic procedures, some cases remain elusive and unclear abdominal pain (UAP) is not infrequent. Emerging evidence supports the hypothesis that functional pain might be attributed to vitamin D deficiency (VDD). People with darker or covered skin are predisposed to developing VDD. Patients in Switzerland stemming from low- and middle-income countries (LMIC) are such a population. Aim: To identify cases with UAP in LMIC patients and to compare vitamin D status with a control group. Methods: A retrospective single-center case-control study was carried out from 1 January 2013 to 31 August 2016 in all adult patients (more than 16 years old) stemming from LMIC and presenting at the university ED of Bern with abdominal pain. Vitamin D status was retrieved from these cases when available. The control group consisted of patients without abdominal pain or metabolic diseases and was matched (1:1) to the cases for age, gender, body mass index, geographic distribution, and season of vitamin D estimation. Results: A total of 10,308 cases from LMIC were reported to the ED. In total, 223 cases were identified with UAP. The status of vitamin D was available for 27 patients; 27 matched individuals were subsequently retrieved for the control group. Women made up 56.7% of the UAP group and 43.3% of the control group. The most common origin of the LMIC subjects was southern Europe (20.4%), followed by southern Asia (16.7%) and Eastern Europe (13%). Fourteen UAP patients exhibited severe VDD (< 25 nmol/L) versus one in the control group (p = 0.001). The difference remained significant if the patients were identified as having VDD (<50 nmol/L) or not (p = 0.024). Comparison of the means indicated that the UAP group had lower vitamin D levels than the control group (41.3 vs. 53.7 nmol/L, respectively), but this difference was marginal (p = 0.060) and not statistically significant. After adjustment for potential confounders, including gender, mean vitamin D levels remained non-significantly different between groups. In the sub-group analysis, vitamin D levels were lower in women than in men (p = 0.037), compared to the respective controls. Conclusion: This study showed for the first time that patients from LMIC who presented to ED with UAP displayed VDD. Validation from larger studies is warranted to evaluate the linkage of VDD with UAP.