Structural and functional implications of the phospholamban hinge domain: impaired SR Ca2+ uptake as a primary cause of heart failure

OBJECTIVE: The role of sarcoplasmic reticulum (SR) in the onset and progression of heart failure is controversial. We tested the hypothesis that impairment of SR Ca2+ sequestration may be a primary cause for progressive left ventricular (LV) dysfunction and the phospholamban hinge domain may be critical in this process. METHODS: A phospholamban hinge domain mutant (PLB/N27A) was introduced in the cardiac compartment of the phospholamban null mouse. An integrative approach was used to characterize the resulting cardiac phenotype at a structural, cellular, whole organ and intact animal level. RESULTS: NMR analysis revealed a defined alteration in the alpha-helical configuration between residues Q22 to F35 in mutant phospholamban. Transgenic lines expressing similar levels of mutant compared to wild-type phospholamban exhibited super-inhibition of the SR Ca2+ ATPase affinity for Ca2+ (EC50 0.52 microM) in oxalate-supported Ca2+ uptake measurements, which translated into impaired relaxation and attenuated responses to beta-adrenergic stimulation. Importantly, a blunted force-frequency relation was observed in mutant hearts preceding left ventricular dilation. Upon aging to 10 months, the predominantly diastolic dysfunction progressed to congestive heart failure, characterized by induction of a fetal gene program, cardiac remodeling, lung congestion, depressed systolic function and early mortality. CONCLUSION: Increased inhibition of Ca2+ sequestration may be a causative factor in the development of left ventricular dysfunction and myocyte remodeling leading to heart failure. Furthermore, the hinge domain may play an important role in transmitting PLB's regulatory effects on SERCA.     

Pica and the elephant's ear

This is a case report of an otherwise healthy 2-year-old boy with a history of pica, associated with iron deficiency anemia. This boy was referred to our department for a neurologic evaluation because of an acute episode of sialorrhea, difficulty in speaking, dysphagia, and repeated swallowing movements. An uncertain episode of a brief-duration still gaze was also reported. In addition, the history revealed that the child had earlier ingested a leaf from a poisonous houseplant called Colocasia esculenta, also known as "elephant's ear." The habit of pica subsided after treatment with iron supplements. A 9-month follow-up period was uneventful. Neurologic manifestations can accompany accidental intoxications of some non-nutrient substances. Thus, pica must be suspected in children with acute behavior alterations.     

Piriformis muscle syndrome: A cross-sectional imaging study in 116 patients and evaluation of therapeutic outcome

Objectives

To increase the clinical awareness of piriformis muscle syndrome (PMs) by reporting cross-sectional imaging findings, the clinical impact of imaging studies and treatment outcome.

Methods

Within a 10-year-period, 116 patients referred for radiological evaluation of clinically suspected PMs, with excluded lumbar pathology related to symptomatology, were prospectively studied with MRI and/or computed tomography (CT). Piriformis muscle (PM), sciatic nerve (SN), piriformis region and sacroiliac joints were evaluated. PMs was categorised into primary/secondary, according to a reported classification system. Treatment decisions were recorded. Outcome was categorised using a 3-point-scale.

Results

Seventy-four patients (63.8%) exhibited pathologies related to PMs. Primary causes were detected in 12 and secondary in 62 patients. PM enlargement was found in 45.9% of patients, abnormal PM signal intensity/density in 40.5% and sciatic neuritis in 25.7%. Space-occupying lesions represented the most common related pathology. Treatment proved effective in 5/8 patients with primary and 34/51 patients with secondary PMs. In 34 patients, imaging revealed an unknown underlying medical condition and altered treatment planning.

Conclusions

Secondary PMs aetiologies appear to prevail. In suspected PMs, PM enlargement represented the most common imaging finding and space-occupying lesions the leading cause. Imaging had the potential to alter treatment decisions.

Key Points

? In clinically suspected PMs cross-sectional imaging may reveal variable pathology. ? Secondary PMs aetiologies appeared to be more common than primary. ? PM enlargement represented the most common imaging finding in clinically suspected PMs. ? Space-occupying lesions in the piriformis region represented the leading cause of PMs. ? In clinically suspected PMs cross-sectional imaging may alter treatment planning.

     

Breast Disorders in Adolescence: A Review of the Literature

BackgroundAdolescence is accompanied by a variety of changes in young breast development, which greatly affects the adolescent''s psychology and socialization.SummaryPubMed, EMBASE, and the Cochrane Library were searched for studies relative to epidemiology, clinical characteristics, diagnosis, and management of all breast disorders in adolescence and their consequences. Development disorders are breast asymmetry, breast atrophy, breast hypoplasia, hypomastia, juvenile breast hypertrophy, and tuberous breast. Breast congenital abnormalities include athelia, amastia, accessory breast tissue, polymastia, polythelia, and congenital disorders of nipples. Breast infections are commonly caused from Gram-positive coccus rather than Gram-negative bacteria. Breast abscess occurs when breast infections are not promptly treated. Nipple discharge is caused by a variety of conditions and should be managed carefully. Fibrocystic changes, cysts, and fibroadenomas are the most common benign masses in adolescence. Primary, secondary, or metastatic breast cancer is extremely rare in adolescence. However, clinicians should include breast cancer in the differential diagnosis of a breast mass in adolescence.Key MessagesClinicians should be aware of all breast disorders that may occur in adolescence. Early diagnosis and treatment will result in the reassurance of adolescents and their families without any detrimental effect on their psychology, sexual behavior, and socialization. Adolescents with breast disorders may require a multidisciplinary approach by a pediatrician, a gynecologist specializing in pediatric-adolescent gynecology, a plastic surgeon, and a psychologist for the best management of breast disorders.     

Initiation Codon Scanthrough versus Termination Codon Readthrough Demonstrates Strong Potential for Major Histocompatibility Complex Class I–restricted Cryptic Epitope Expression

Accumulating evidence shows that the repertoire of major histocompatibility complex class I–restricted epitopes extends beyond conventional translation reading frames. Previously, we reported that scanthrough translation, where the initiating AUG of a primary open reading frame is bypassed, is most likely to account for the presentation of cryptic epitopes from alternative reading frames within the influenza A PR/8/34 nucleoprotein gene. Here, we confirm and extend these findings using an epitope cassette construct that features two well-defined CD8⁺ T cell (T_CD8+) epitopes in alternative reading frames, each preceded by a single start codon. Expression of one epitope depends on scanning of the ribosome over the first AUG with translation initiation occurring at the second AUG. We find that scanthrough translation has great potency in our system, with its impact being modulated, as predicted, by the base composition surrounding the first initiation codon, the number of start codons preceding the point of alternate reading frame initiation, and the efficiency with which the epitope itself is generated. Additionally, we investigated the efficiency of eukaryotic translation termination codons, to assess codon readthrough as a mechanism for cryptic epitope expression from 3′ untranslated regions. In contrast with initiation codons, eukaryotic stop codons appear to be highly efficient at preventing expression of epitopes encoded in 3′ untranslated regions, suggesting that 3′ untranslated regions are not a common source of cryptic epitope substrate. We conclude that scanthrough is a powerful mechanism for the expression of epitopes encoded in upstream alternative open reading frames that may contribute significantly to T_CD8+ responses and to tolerance induction.T_CD8+ activation depends upon interaction between the clonotypic TCR and a MHC class I molecule complexed with an 8–10 amino acid peptide epitope (1–5). Conventional models of antigen processing and presentation generally presume these epitopes to be derived from the cytosolic proteolysis of full-length, fully translated proteins. However, it has been demonstrated that T_CD8+ are also capable of recognizing either the products of gene fragments or mini genes expressing the minimal sequence of an epitope (6–8). In addition, there is sufficient evidence in the literature to suggest that strict adherence to conventional translation is not required for antigen presentation. Examples include the presentation of epitopes encoded in untranslated regions (UTRs)¹ (9, 10) and in alternative reading frames (RF) (11–15). This implies that regions of genes that are traditionally thought to be inaccessible to translation could be a potentially significant source of substrate for antigen processing and presentation.The generally accepted model for translation initiation of most messages is the ribosomal scanning hypothesis (16– 18), where a ribosome scans from the 5′-cap and begins translation from the first AUG in favorable context, as determined by surrounding nucleotide identity. This AUG defines the primary RF, or RF0. Extensive surveys of eukaryotic genes have shown that in most cases the first AUG encountered is in favorable context (19, 20) and biochemical studies have shown that a primary start codon is used by the great majority of scanning ribosomes (21). However, some ribosomes fail to initiate at the primary initiation codon, with factors such as proximity to the 5′-cap, and local secondary structures as well as initiation codon context having influence (22, 23). We have recently demonstrated that initiation codon readthrough (scanthrough) is probably responsible for the production of epitopes encoded in alternative RFs from a mutated influenza A PR/8/34 nucleoprotein (NP) gene (24). In the system studied, it appeared that scanthrough was capable of utilizing an internal AUG codon to rescue the presentation of three NP epitopes (NP_50–57, NP_147–155, NP_366–374) that had been shifted out-of-frame by a base deletion in the second codon of the NP open reading frame (ORF). In addition to providing this alternative source of out-of-frame epitopes, scanthrough translation may simultaneously produce truncated, in-frame polypeptides that could be more efficiently processed than full-length protein by the class I processing machinery. This mechanism could also provide a plausible rationale for the presentation of exocytic proteins that do not normally access the cytosolic protein-degradation apparatus, by allowing initiation of translation after the signal sequence encoding region, resulting in the production of a nontranslocated substrate.As in the case of start codons, not all translation stop codons cause termination of translation with equal efficiency. The sequence surrounding the termination codon dictates whether some readthrough and continued translation can occur. In Saccharomyces cerevisiae, termination codon readthrough has been shown to occur at levels up to 3.35% (25). This mechanism would allow for translation from the 3′ UTR, further expanding the available substrate for class I presentation.In the current study, we have constructed a presentation cassette in order to assess the potential for ribosomal scanthrough in epitope expression under controlled conditions. This allowed us, first, to confirm our previous findings concerning initiation codon readthrough and, second, to assess directly the strength of initiation codon readthrough in the face of competitive AUG codons encoded in alternate RFs. In addition, we wished to discern whether epitopes encoded in 3′ UTR are also available for presentation. For this purpose, we tested the ability of ribosomes to read through strong and weak RF0 stop signals inserted in wild-type NP and translate a downstream epitope. These studies provide important insights into the translation mechanisms available for the production of substrate from unconventional coding regions.     

Informal out-of-pocket payments experience and individuals’ willingness-to-pay for healthcare services in Greece

BackgroundInformal out-of-pocket payments to healthcare providers are not uncommon in the Greek health system. We explore individuals’ willingness-to-pay (WTP) to secure zero out-of-pocket full coverage for healthcare services and medications and we estimate the impact of past informal payments and individuals’ opinion about the legalization of informal payments on WTP.MethodsWe conducted a survey of 2841 participants from November 2016 to February 2017. We obtained information on WTP using the contingent valuation method. A two-part regression model was used to estimate the association between WTP, informal payments, and respondents’ opinion about legalizing such payments.ResultsAbout 80% of the respondents were willing to pay an average of €95 per month to obtain free access to full healthcare coverage and medications. About 65% of the respondents were involved in an informal payment at least once during the past four months with an average payment of €247. Higher informal payments and supportive opinions towards the legalization of informal payments increased the likelihood of WTP and were also positively associated with increased WTP amounts overall (p < 0.001).ConclusionsThis survey reveals that individuals’ WTP is critically affected by previous experiences and attitudes towards informal payments. Our results imply that the potential introduction of official fees might not suffice to limit informal payments and suggest the need for stricter regulatory policies.     

Medical personnel and patient dosimetry during coronary angiography and intervention

Percutaneous coronary interventions are associated with increased radiation exposure compared to most radiological examinations. This prospective study aimed at (1) measuring entrance doses for all in-room personnel, (2) performing an assessment of patient effective dose and intracoronary doses, (3) investigating the contribution of each projection to kerma-area product (KAP) and irradiation time, (4) comparing results with established DRL values in this clinical setting and (5) estimating the risk for fatal cancer to patients and operators. Measurements were performed during 40 consecutive procedures of coronary angiography (CA), half of which were followed by ad hoc coronary angioplasty (PTCA). KAP measurements were used for patients and thermoluminescent dosimetry for the in-room personnel. The mean KAP value per procedure for CA was 29 +/- 9 Gy cm2. Thirty four per cent of KAP was due to fluoroscopy, whereas the remainder (66%) was due to digital cine. Accordingly, the mean KAP value per PTCA procedure was 75 +/- 30 Gy cm2, and contribution of fluoroscopy is 57%. Effective dose per year was estimated to be 0.04-0.05 mSv y(-1) for the primary operator, and 0.03-0.04 mSv y(-1) for those assisting. Corresponding measurements for radiographer and nurse were below detectable level, implying minimal radiation hazards for them. Regarding radiation exposure, coronary intervention is considered a quite safe procedure for both patients and personnel in laboratories with modern equipment and experienced operators as long as standard safety precautions are considered. Exposure optimization though should be constantly sought through continuous review of procedures.     

Distribution of CD1A-positive langerhans cells and lymphocyte subsets in transitional cell carcinoma of the urinary bladder. An immunohistological study on frozen sections

The distribution of CD1a-positive Langerhans cells, CD4-positive T-helper cells, and CD8-positive T-suppressor cells in 36 patients with transitional cell carcinoma of the urinary bladder was studied immunohistochemically on frozen sections. Multiple tissue specimens from the tumour, the adjacent mucosa, and random bladder wall biopsies were examined. Langerhans cells were mainly interspersed among the tumour cells, whereas T-helper cells were present in aggregates in the stroma. T-suppressor cells were present both in aggregates in the stroma and among the tumour cells. There was a marginal relationship between the density of Langerhans cells and the density of T-helper/inducer cells and a good relationship with CD8-positive cells. There was no statistically significant difference in the population density of Langerhans cells associated with the various clinicopathological variables, including growth pattern, histological grade and stage, or patient's age and sex. On the contrary, a statistically significant difference was found in the CD1a/CD4 ratio among specimens of different grades. These results show that CD1a cell populations correlate with T-cell populations in bladder cancer, suggesting that Langerhans cells take part in the immune response carried out by T lymphocytes, their task being apparently antigen presentation.