RNA-based recognition and targeting: sowing the seeds of specificity.

RNA is involved in the regulation of multiple cellular processes, often by forming sequence-specific base pairs with cellular RNA or DNA targets that must be identified among the large number of nucleic acids in a cell. Several RNA-based regulatory systems in eukaryotes, bacteria and archaea, including microRNAs (miRNAs), small interfering RNAs (siRNAs), CRISPR RNAs (crRNAs) and small RNAs (sRNAs) that are dependent on the RNA chaperone protein Hfq, achieve specificity using similar strategies. Central to their function is the presentation of short 'seed sequences' within a ribonucleoprotein complex to facilitate the search for and recognition of targets.

LifeTime and improving European healthcare through cell-based interceptive medicine.

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

Bacterial RNA Biology on a Genome Scale.

Bacteria are an exceedingly diverse group of organisms whose molecular exploration is experiencing a renaissance. While the classical view of bacterial gene expression was relatively simple, the emerging view is more complex, encompassing extensive post-transcriptional control involving riboswitches, RNA thermometers, and regulatory small RNAs (sRNAs) associated with the RNA-binding proteins CsrA, Hfq, and ProQ, as well as CRISPR/Cas systems that are programmed by RNAs. Moreover, increasing interest in members of the human microbiota and environmental microbial communities has highlighted the importance of understudied bacterial species with largely unknown transcriptome structures and RNA-based control mechanisms. Collectively, this creates a need for global RNA biology approaches that can rapidly and comprehensively analyze the RNA composition of a bacterium of interest. We review such approaches with a focus on RNA-seq as a versatile tool to investigate the different layers of gene expression in which RNA is made, processed, regulated, modified, translated, and turned over.

Perceptions of clinical teachers acting as examiners regarding the value of Objective Structured Clinical Examinations.

OBJECTIVES: OSCE (Objective Structured Clinical Examination) is a common method of assessing clinical skills used at many universities. An important and at the same time difficult aspect of good examination preparation is obtaining a properly trained and well-motivated group of assessors. To effectively recruit and maintain cooperation with assessors, it is worth to know their opinion. The aim of this study was to investigate the opinions of teacher-examiners about OSCE and to identify the factors that could shape this opinion and influence on motivation. METHODS: A cross-sectional study was conducted using a questionnaire on teachers who participated as OSCE examiners. This questionnaire consisted of 21 questions about their perceptions. Answers were rated in a five-point Likert-type scale. Chi-square or Fisher's exact test was used to analyze the data. RESULTS: A total of 49 (out of 52) teachers participated in this study. Nearly 90% of examiners believed that it is fair, more than 90% that it is transparent. Despite the fact that 67% of examiners believe the examination is difficult to organize and 71% believe it is stressful for students, according to 72% of respondents the OSCE has a positive effect on learning. More than 91% of examiners believed that the OSCE is an appropriate test to assess students' skills. Opinions about examination were independent of specialty, seniority, gender or taking the OSCE as students. CONCLUSION: Teacher-examiners viewed the OSCE as a fair and transparent examination, adequate for assessment of skills and, despite it being difficult to organize, worth doing as it is appropriate to assess practical skills and positively influences students' motivation to learn tested skills.

Clinical communication course and other factors affecting patient-centered attitudes among medical students.

OBJECTIVES: Patient-centered care (PCC) is associated with better relationships, resulting in a decrease in symptoms, hospitalizations and health costs. However, studies analyzing factors influencing patient-centered attitudes show ambiguous results. The purpose was to assess the impact of the Clinical Communication Course (CCC) in Jagiellonian University, Cracow and other factors on Patient-Centered Attitudes (PCA) and Attitude toward Clinical Skills Learning (CSLA). METHODS: We retrospectively compared Polish-speakers (CCC+, n = 160), English-speakers (CCCen+, n = 55) after the CCC and upperclassmen Polish-speakers without it (CCC-, n = 122). Validated questionnaires to measure PCA (Leeds Attitude Toward Concordance II and Patient-Practitioner Orientation Scale (PPOS)) and for CSLA (Communication Skills Attitude Scale with negative subscale (CSAS-N)) were used. The higher the scores, the more PCA, and negative CSLA respectively. Students completed questionnaires and answered questions regarding age, sex, motivation to study (coded as humanitarian - MotHUM, financial - MotFIN, combination - MotMIX) and considered specialization - coded as with more human contact (family medicine, psychiatry, pediatrics - SpecHUM) and others (SpecNHUM). Statistics were prepared in R. RESULTS: CCC+ scored higher in PPOS (2.91 vs. 2.74; p = 0.003) than CCC- and higher in CSAS-N than CCCen+ (31.22 vs. 28.32; p = 0.004). In CCC+ SpecHUM scored lower than SpecNHUM in PPOS (2.65 vs. 2.94, p = 0.012). MotFIN scored higher then MotMIX in PPOS (3.01 vs. 2.7, p = 0.036). Correlations were statistically significant. CONCLUSION: CCC improved PCA in CCC+. They showed more negative CSLA than CCCen+. Among CCC+, surprisingly, SpecNHUM presented more PCA than SpecHUM as well as MotFIN compared to MotMIX.

Identification of Seniors at Risk scale as a simple tool of elderly patients' assessment in an acute hospital department.

INTRODUCTION: e aim of the study was to evaluate the usefulness of Identification of Seniors at Risk (ISAR) scale among elderly patients admitted to the department of internal medicine. MATERIAL AND METHODS: The ISAR score was performed among patients aged >59 years a er admission to the hospital ward. Data from medical history about diseases, taken medicines, falls, length of hospital stay and mortality were compared in patients with ISAR score of 0-1 and over 1 and in subjects with and without history of falls. Regression analysis was used to detect predictors of the length of hospital stay or death. RESULTS: The sample consisted of 102 subjects aged 80.9 ± 7.9 years, 45.5% of men, 34.6% had history of falls. The number of diseases was 11.3 ± 3.9 and number of medicines - 8.9 ± 3.7. The score of ISAR ≥2 was found in 90.2% of patients, length of hospital stay was 10.3 ± 8.4 days, mortality rate was 9.9%. Patients with ISAR score <2 were younger, showed a smaller number of diseases, used less drugs and had less frequency of falls than those with score ≥2. Patients with history of falls had higher mean ISAR score, higher number of diseases and medicines than the others. The increased number of diseases and higher ISAR score significantly influenced the length of hospital stay. None of the analyzed factors had any impact on mortality. CONCLUSION: The score of ISAR scale together with number of diseases have a positive impact on the length of hospital stay.

The Objective Structured Clinical Examination (OSCE) from the perspective of 3rd year's medical students - a pilot study.

BACKGROUND: In 2015 Department of Medical Education in Medical College of Jagiellonian University in Cracow performed a new format of integrated multidisciplinary skills assessment of third year students of medicine a er completing initial courses in internal medicine, surgery, pediatrics and gynecology. AIM: OSCE assessed from the perspective of 3rd year medical students. METHODS: Students of 3rd year's study of Faculty of medicine were evaluated by OSCE, which assessed their internal medicine, surgery, gynecology, pediatric skills. OSCE consisted of 12 stations. In order to assess student's opinions on OSCE, we used method of diagnostic survey; Statistica 12.0. RESULTS: OSCE passed 255 (98.83%) of the students in thefirst term. We analyzed 221 questionnaires, in which students expressed their opinion. 93.7% of the students considered OSCE as a well organized exam. 87.8% of students claimed that OSCE is fair and 95.5% that the exam is clear. 86.4% students are pleased with the introduction information about OSCE that was given before the examination. 78.6% students believe, that OSCE allows to properly identify skills that require improvement. Students, who didn't pass all stations with positive result, more o en (Chi^2 Pearsona, p = 0.01990) indicated improper balance between quantity of stations that check communication skills and these checking other clinical skills. CONCLUSIONS: is study confirms that OSCE in the students' opinions was well organized and fair. It proves that OSCE correctly selects students who need to improve their knowledge and skills. Students' preparing to the exams, their knowledge and skills, influences on the students' perception of OSCE.

Modulation of RNA polymerase assembly dynamics in transcriptional regulation.

The interaction of transcription factors with target genes is highly dynamic. Whether the dynamic nature of these interactions is merely an intrinsic property of transcription factors or serves a regulatory role is unknown. Here we have used single-cell fluorescence imaging combined with computational modeling and chromatin immunoprecipitation to analyze transcription complex dynamics in gene regulation during the cell cycle in living cells. We demonstrate a link between the dynamics of RNA polymerase I (RNA Pol I) assembly and transcriptional output. We show that transcriptional upregulation is accompanied by prolonged retention of RNA Pol I components at the promoter, resulting in longer promoter dwell time, and an increase in the steady-state population of assembling polymerase. As a consequence, polymerase assembly efficiency and, ultimately, the rate of entry into processive elongation are elevated. Our results show that regulation of rDNA transcription in vivo occurs via modulation of the efficiency of transcription complex subunit capture and assembly.

Single-cell RNA-seq: advances and future challenges.

Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Single-cell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNA-seq protocols have been introduced and are reviewed here-each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.

Prognostic factors of left ventricle function deterioration in patients with coronary disease and normal results of echocardiographic examination in a 2-year observation - prospective cohort study.

INTRODUCTION: Coronary disease is one of the strongest risk factors of developing heart failure. Identification of the predictive factors of left ventricle (LV) function deterioration in patients with stable angina pectoris and normal systolic function of LV, is a challenge for clinicians. OBJECTIVE: Identification of prognostic factors: clinical, echocardiographic, biochemical (NT-proBNP, hsCRP) and spiroergometric of left ventricle function deterioration in patients with coronary disease and normal results of baseline echocardiographic examination in a 2-year observation. PATIENTS AND METHODS: The study group comprised of 32 patients with stable angina pectoris and normal heart function, aged 50.9 ± 4 years; 23 men (71.8%). At baseline we performed echocardiography, cardiopulmonary exercise testing and determined serum levels of B-type natriuretic peptide and C-reactive protein. 24 months later we performed echocardiography and cardiopulmonary exercise testing. RESULTS: Patients with stable angina pectoris and normal LV function are at risk of developing the impairment of LV function. Diastolic dysfunctions of LV are a crucial element of the whole clinical picture. 53.1% developed of LV diastolic dysfunction: 37% isovolumetric relaxation disorders and 15% pseudonormalization. The analysis of the tests carried out after a 2-year observation indicated a relation-ship between developing diastolic dysfunction of LV and the presence of type II diabetes (p = 0.01). Peak oxygen consumption (VO2peak) at baseline was significantly lower in patients who developed diastolic dysfunction of LV a er 2 years (p = 0.03). CONCLUSIONS: The predictors of LV diastolic function deterioration in a 2-year observation in this group of patients include type II diabetes and peak oxygen consumption VO2peak.

Prognostic thresholds of outcome predictors in severe accidental hypothermia.

Hemodynamically unstable patients with severe hypothermia and preserved circulation should be transported to dedicated extracorporeal life support (ECLS) centers, but not all are eligible for extracorporeal therapy. In this group of patients, the outcome of rewarming may sometimes be unfavorable. It is, therefore, crucial to identify potential risk factors for death. Furthermore, it is unclear what criterion for hemodynamic stability should be adopted for patients with severe hypothermia. The aim of this study is to identify pre-rewarming predictors of death and their threshold values in hypothermic patients with core temperature ≤ 28 °C and preserved circulation, who were treated without extracorporeal rewarming. We conducted a multicenter retrospective study involving patients in accidental hypothermia with core temperature 28 °C or lower, and preserved spontaneous circulation on rewarming initiation. The data were collected from the International Hypothermia Registry, HELP Registry, and additional hospital data. The primary outcome was survival to hospital discharge. We conducted a multivariable logistic regression and receiver operating characteristic curve (ROC) analysis. In the multivariate analysis of laboratory tests and vital signs, systolic blood pressure (SBP) adjusted for cooling circumstances and base excess (BE) were identified as the best predictor of death (OR 0.974 95% CI 0.952-0.996), AUC ROC 0.79 (0.70-0.88). The clinically relevant cutoff for SBP was identified at 90 mmHg with a sensitivity of 0.74 (0.54-0.89) and a specificity of 0.70 (0.60-0.79). The increased risk of death among hypothermic patients with preserved circulation occurs among those with an SBP below 90 mmHg and in those who developed hypothermia in their homes.

The potential of virtual triage AI to improve early detection, care acuity alignment, and emergent care referral of life-threatening conditions.

Objective: To evaluate the extent to which patient-users reporting symptoms of five severe/acute conditions requiring emergency care to an AI-based virtual triage (VT) engine had no intention to get such care, and whose acuity perception was misaligned or decoupled from actual risk of life-threatening symptoms. Methods: A dataset of 3,022,882 VT interviews conducted over 16 months was evaluated to quantify and describe patient-users reporting symptoms of five potentially life-threatening conditions whose pre-triage healthcare intention was other than seeking urgent care, including myocardial infarction, stroke, asthma exacerbation, pneumonia, and pulmonary embolism. Results: Healthcare intent data was obtained for 12,101 VT patient-user interviews. Across all five conditions a weighted mean of 38.5% of individuals whose VT indicated a condition requiring emergency care had no pre-triage intent to consult a physician. Furthermore, 61.5% intending to possibly consult a physician had no intent to seek emergency medical care. After adjustment for 13% VT safety over-triage/referral to ED, a weighted mean of 33.5% of patient-users had no intent to seek professional care, and 53.5% had no intent to seek emergency care. Conclusion: AI-based VT may offer a vehicle for early detection and care acuity alignment of severe evolving pathology by engaging patients who believe their symptoms are not serious, and for accelerating care referral and delivery for life-threatening conditions where patient misunderstanding of risk, or indecision, causes care delay. A next step will be clinical confirmation that when decoupling of patient care intent from emergent care need occurs, VT can influence patient behavior to accelerate care engagement and/or emergency care dispatch and treatment to improve clinical outcomes.

The road much traveled: trafficking in the cell nucleus.

Trafficking of RNA molecules and proteins within the cell nucleus is central to genome function. Recent work has revealed the nature of RNA and protein motion within the nucleus and across the nuclear membrane. These studies have given insight into how molecules find their destinations within the nucleus and have uncovered some of the structural properties of the nuclear microenvironment. Control of RNA and protein trafficking is now emerging as a physiological regulatory mechanism in gene expression and nuclear function.

The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks.

DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of transcription-coupled repair enzymes, and inhibition of RNA synthesis. During the S phase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication. Whether the transcription machinery is affected by the presence of DNA double-strand breaks remains a long-standing question. Here we monitor RNA polymerase I (Pol I) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in Pol I transcription. Surprisingly, we find Pol I inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with Pol I initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks.

The association between quality of connections and diagnostic accuracy in student-generated concept maps for clinical reasoning education with virtual patients.

Objectives: Concept maps are a learning tool that fosters clinical reasoning skills in healthcare education. They can be developed by students in combination with virtual patients to create a visual representation of the clinical reasoning process while solving a case. However, in order to optimize feedback, there is a need to better understand the role of connections between concepts in student-generated maps. Therefore, in this study we investigated whether the quality of these connections is indicative of diagnostic accuracy. Methods: We analyzed 40 concept maps created by fifth-year medical students in the context of four virtual patients with commonly encountered diagnoses. Half of the maps were created by students who made a correct diagnosis on the first attempt; the other half were created by students who made an error in their first diagnosis. The connections in the maps were rated by two reviewers using a relational scoring system. Analysis of covariance was employed to examine the difference in mean connection scores among groups while controlling for the number of connections. Results: There were no differences between the groups in the number of concepts or connections in the maps; however, maps made by students who made a correct first diagnosis had higher scores for the quality of connections than those created by students who made an incorrect first diagnosis (12.13 vs 9.09; p=0.03). We also observed students' general reluctance to use connections in their concept maps. Conclusion: Our results suggest that the quality, not the quantity, of connections in concept maps is indicative of their diagnostic accuracy.

Extracorporeal Rewarming Is Associated With Increased Survival Rate in Severely Hypothermic Patients With Preserved Spontaneous Circulation.

Treatment recommendations for rewarming patients in severe accidental hypothermia with preserved spontaneous circulation have a weak evidence due to the absence of randomized clinical trials. We aimed to compare the outcomes of extracorporeal versus less-invasive rewarming of severely hypothermic patients with preserved spontaneous circulation. We conducted a multicenter retrospective study. The patient population was compiled based on data from the HELP Registry, the International Hypothermia Registry, and a literature review. Adult patients with a core temperature <28°C and preserved spontaneous circulation were included. Patients who underwent extracorporeal rewarming were compared with patients rewarmed with less-invasive methods, using a matched-pair analysis. The study population consisted of 50 patients rewarmed extracorporeally and 85 patients rewarmed with other, less-invasive methods. Variables significantly associated with survival included: lower age; outdoor cooling circumstances; higher blood pressure; higher PaCO 2 ; higher BE; higher HCO 3 ; and the absence of comorbidities. The survival rate was higher in patients rewarmed extracorporeally ( p = 0.049). The relative risk of death was twice as high in patients rewarmed less invasively. Based on our data, we conclude that patients in severe accidental hypothermia with circulatory instability can benefit from extracorporeal rewarming without an increased risk of complications.

The Use of Simulated Patients Is more Effective than Student Role Playing in Fostering Patient-Centred Attitudes during Communication Skills Training: A Mixed Method Study.

Objective: While simulated patients (SPs) are considered a standard tool in communication skills training, there is no evidence thus far of their comparative benefit to the more cost-effective option of student role playing. We compared the effectiveness of both approaches in developing patient-centred attitudes in students. Methods: We retrospectively compared students who participated in the clinical communication course (CCC), based on student role playing (CCCsp-, n = 160), to students who participated in the CCC with SPs (CCCsp+, n = 146), and students with no formal CCC (CCC-, n = 122). We used validated questionnaires to assess patient centredness. We also conducted focus group interviews (FGI) to better understand the impact of CCC with sp. Results: Students after the CCC with simulated patients achieved a significantly higher score in the patient-practitioner orientation scale than other groups (p < 0.001). Conclusions: There is a strong positive correlation between the implementation of simulated patients and patient-centred attitudes among students. Data from the FGI revealed that students perceived training with SP as more realistic, safe, and engaging than student role playing. Practice Implications. Our research provides evidence to justify costs and resources invested in simulated patient programs.

[Nailfold videocapillaroscopy - a useful tool for screening patients with juvenile idiopathic arthritis at the risk of development of premature atherosclerosis]. / Wideokapilaroskopia lozyska okolopaznokciowego - uzyteczne narzedzie w diagnostyce pacjentów z mlodzienczym idiopatycznym zapaleniem stawów zagrozonych rozwojem przedwczesnych zmian miazdzycowych.

Inflammation of the vascular wall with endothelial dysfunction and subsequent activation of inflammatory immune response play pivotal roles in the early development of the atherosclerotic process not only in adults with rheumatoid arthritis (RA), but also in children with juvenile idiopathic arthritis (JIA). This hypothesis was supported by our findings from autopsy examination, revealing atherosclerosis lesions in about 30% children with JIA. The established methods of assessing pre-clinical atherosclerosis include measurement of biochemical markers of endothelium impairment and ultrasonographic examination of vessels (FMD, IMT). The authors suggest that revealing structural and functional impairment of peripheral microvessels by means of static and dynamic videocapillaroscopy can give clinicians a chance to identify even younger patients with JIA/RA at high risk of atherosclerosis.