Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation.

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.

PRC1 sustains the integrity of neural fate in the absence of PRC2 function.

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We found that PRC1 is essential for the specification of segmentally restricted spinal motor neuron (MN) subtypes, while PRC2 activity is dispensable to maintain MN positional identities during terminal differentiation. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox-dependent specification programs by derepressed Hox13 paralogs (Hoxa13, Hoxb13, Hoxc13, Hoxd13). These results indicate that PRC1 can function in the absence of de novo PRC2-dependent histone methylation to maintain chromatin topology and postmitotic neuronal fate.

De novo assembly and delivery to mouse cells of a 101 kb functional human gene.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

Differential abilities to engage inaccessible chromatin diversify vertebrate Hox binding patterns.

Although Hox genes encode for conserved transcription factors (TFs), they are further divided into anterior, central and posterior groups based on their DNA-binding domain similarity. The posterior Hox group expanded in the deuterostome clade and patterns caudal and distal structures. We aimed to address how similar Hox TFs diverge to induce different positional identities. We studied Hox TF DNA-binding and regulatory activity during an in vitro motor neuron differentiation system that recapitulates embryonic development. We found diversity in the genomic binding profiles of different Hox TFs, even among the posterior group paralogs that share similar DNA-binding domains. These differences in genomic binding were explained by differing abilities to bind to previously inaccessible sites. For example, the posterior group HOXC9 had a greater ability to bind occluded sites than the posterior HOXC10, producing different binding patterns and driving differential gene expression programs. From these results, we propose that the differential abilities of posterior Hox TFs to bind to previously inaccessible chromatin drive patterning diversification.This article has an associated 'The people behind the papers' interview.

Prevalence of diabetes mellitus (DM) in tuberculosis (TB) patients: clinical and radiologic features in the TB-DM association based on a five-year hospital study.

A previously known association of tuberculosis (TB), now a global emergency, with diabetes mellitus (DM), a chronic disease of increasing prevalence worldwide, has resurfaced. DM is a recognised TB risk factor and M. tuberculosis infection usually disturbs glycoregulation. We aimed to estimate DM prevalence among TB patients and to analyse clinical and radiologic manifestations of TB in this group. The cross-sectional study included 1017 patients discharged with a TB diagnosis from a tertiary level facility in a five-year period. After exclusion of 128 patients with TB sequelae, we selected 889 patients with confirmed active TB, and the final selection led to 88 subjects with both active TB and DM. DM was found in 9.89% of active TB patients. Testing hypotheses for single-sample proportions showed that the prevalence was significantly higher than the prevalence in the general population (p<0.01). The average patient age was 60.47±14.88 years (range: 20-88), while the male/female ratio was 2.26. We found pulmonary TB in 96.3% of the 88 subjects, and extrapulmonary in 3.4%, the latter more frequently in women (p=0.08). Cavities were more frequently found in tobacco smokers compared to non-smokers (p=0.002) and in patients living in rural settings (p=0.002); haemoptysis was detected more frequently in men compared to women (p=0.044). Half of the patients had no fever at all, and only 14.8% had tachycardia. Auscultatory findings were positive in 57 (64.8%) patients. Radiographic changes were atypical in 17/88 (19.3%) patients and included pulmonary bases in seven patients, upper lobe anterior segment, and disseminated miliary pattern in two patients. DM prevalence is significantly higher among TB patients compared to the general population. The mean age of TB patients with DM is significantly higher than expected in TB patients alone. Knowledge of TB clinical and radiologic variations in this group of patients may reduce the delay in TB diagnosis with its clinical and epidemiologic consequences.

CTCF-mediated topological boundaries during development foster appropriate gene regulation.

The genome is organized into repeating topologically associated domains (TADs), each of which is spatially isolated from its neighbor by poorly understood boundary elements thought to be conserved across cell types. Here, we show that deletion of CTCF (CCCTC-binding factor)-binding sites at TAD and sub-TAD topological boundaries that form within the HoxA and HoxC clusters during differentiation not only disturbs local chromatin domain organization and regulatory interactions but also results in homeotic transformations typical of Hox gene misregulation. Moreover, our data suggest that CTCF-dependent boundary function can be modulated by competing forces, such as the self-assembly of polycomb domains within the nucleus. Therefore, CTCF boundaries are not merely static structural components of the genome but instead are locally dynamic regulatory structures that control gene expression during development.

Illness perception in tuberculosis by implementation of the Brief Illness Perception Questionnaire - a TBNET study.

How patients relate to the experience of their illness has a direct impact over their behavior. We aimed to assess illness perception in patients with pulmonary tuberculosis (TB) by means of the Brief Illness Perception Questionnaire (BIPQ) in correlation with patients' demographic features and clinical TB score. Our observational questionnaire based study included series of consecutive TB patients enrolled in several countries from October 2008 to January 2011 with 167 valid questionnaires analyzed. Each BIPQ item assessed one dimension of illness perceptions like the consequences, timeline, personal control, treatment control, identity, coherence, emotional representation and concern. An open question referred to the main causes of TB in each patient's opinion. The over-all BIPQ score (36.25 ± 11.054) was in concordance with the clinical TB score (p ≤ 0.001). TB patients believed in the treatment (the highest item-related score for treatment control) but were unsure about the illness identity. Illness understanding and the clinical TB score were negatively correlated (p < 0.01). Only 25% of the participants stated bacteria or TB contact as the first ranked cause of the illness. For routine clinical practice implementation of the BIPQ is convenient for obtaining fast and easy assessment of illness perception with potential utility in intervention design. This time saving effective personalized approach may improve communication with TB patients and contribute to better behavioral strategies in disease control.

Giving blood donors something to drink before donation can prevent fainting symptoms: is there a physiological or psychological reason?

The vasovagal reaction has been widely studied but its anatomic and physiological nature remains uncertain. The mechanisms underlying vasovagal reaction related to blood donation are not completely understood either. Does its occurrence depend on the blood donors' physical characteristics and health variables or psychological factors? On the basis that a psychological approach considerably prevents donor reactions, the effect of fruit juice ingestion was studied in a group of 1849 first-time high-school students as a simple strategy to avoid systemic reactions at blood donation. The reasons for the psychological effect of this hydration protocol are stressed also in light of previous physiological studies on the hemodynamic effects of water or carbohydrate drinks.

Gender differences revealed by the Brief Illness Perception Questionnaire in allergic rhinitis.

BACKGROUND: The increasing prevalence of allergic rhinitis (AR) is reported worldwide. Illness perception (IP) assessment is warranted in current routine clinical practice to assist communication between patients and medical staff, and improve adherence to treatment and disease outcome. OBJECTIVE: To investigate a group of patients with AR in terms of their IP by the Brief Illness Perception Questionnaire (BIPQ) and to correlate the findings with demographic and clinical features. METHODS: In this observational questionnaire-based study, a successive series of patients treated for AR at the Allergology and Immunology Teaching Hospital, Clinical Centre of Serbia in Belgrade, were enrolled from September 2010 to January 2011, and 93 valid questionnaires were analyzed. Each item of the BIPQ assessed one dimension of IP like the consequences, timeline, personal control, treatment control, identity, coherence, emotional representation and concern. RESULTS: The patients' average age: 35.25 ± 12.42; male/female ratio: 0.79; the overall BIPQ score = 34.69 ± 11.89. The highest item-related scores were found for treatment control (8.17 ± 2.28), illness understanding (7.34 ± 2.96) and emotional representation (6.30 ± 3.45), and the lowest for identity (4.8 ± 2.78) and affection (4.83 ± 2.65). Women compared with men perceive AR as a significantly more threatening disease (P = 0.04). No significant correlation between the BIPQ total or item-related scores was found for any other demographic or clinical feature. CONCLUSION: The BIPQ, which allows rapid assessment of IP and reveals gender differences in AR, is a convenient tool for use in routine clinical practice. Further investigation is needed to demonstrate how IP may influence patients' behavior in AR, treatment adherence and disease outcome.

Emotional support to apheresis donors: effect and implication.

It is widely accepted that well-being and satisfaction are unavoidably interlinked requirements that predispose donors to become regular. Detecting in advance even the mildest symptoms of discomfort during first donation, that may simply be the result of an unknown experience, is useful in preventing more disturbing symptoms (mens sana in corpore sano). Being able to recognize any form of uneasiness, even when not immediately apparent, creates in donors the awareness that staff is professionally skilled and really interested in their safety and comfort. This reinforces the staff/donor relationship and gives donors the chance to keep their calm and to learn how to manage disturbing emotions. Emotional support given to first-time platelet familiar donors during donation prevented or mitigated the onset of vaso-vagal symptoms and also freed positive emotions that reinforced the desire to donate. As a result, 62% of these donors, informed about the shortage of voluntary platelet donors, spontaneously gave their address contact to be called also for non-familiar recipients. The application of the psychological approach in Italy increased the rate of donor return to 97% and its partial application in Serbia was useful in converting familiar donors into voluntary ones.

Blood donor satisfaction and the weak link in the chain of donation process.

Recruiting and retaining enough donors is a problem. Six hundred and thirty nine blood donors were interviewed to identify the elements that can influence their satisfaction and the decision to become regulars. The majority of them were satisfied with staff skill and communication, they declared they were calm before donating and felt well after donation. First-time donors (FTDs) were more frightened, showed anticipatory anxiety, were less sure they would donate again (p<0.01) and the youngest were also less satisfied with the staff's behavior after donation. Identifying the more scared FTDs and diminishing their anxiety before donating could positively influence their decision to become regulars.

Knowledge and attitudes towards tuberculosis in non medical students University of Belgrade.

BACKGROUND: Population's knowledge on tuberculosis (TB) is crucial in early seeking of medical care. Delay in diagnosis for any reason contributes to advanced forms and TB transmission in the community. Knowledge about TB in general population of Serbia is poor, including vulnerable groups. AIM: to assess knowledge about TB in a group of non medical students in University of Belgrade, their attitudes towards TB patients, sources of medical information they use or desire. METHODS: Observational, questionnaire based study. University students of the Faculty of Organizational Science and Faculty of Geography completed the 27-item questionnaires voluntarily and anonymously. The questions related to TB etiology, way of transmission, risk factors and the source of health information students preffer. Statistical analysis was performed. RESULTS: All the participants (69 students aged 20 +/- 0.777 years, 69.7% male, 30.3% female) previously heard about TB, mostly describing it as pulmonary disease. Only 22 (31.88%) selected bacillus as the only cause of TB, and 20% selected answer "I do not know". TB is curable (95%). One third does not know about TB symptoms and 45/69 (65%) think that TB is an infectious disease. Majority (78.3%) would pay visit to TB patient, 1/2 with fear of infection that is in positive correlation with knowledge on infectiousness (p=0.041). Television is the most common used source of health information. Students have the greatest confidence in doctors' information. CONCLUSION: Students' knowledge on TB is inadequate, especially on its cause and way of transmission. More effort is needed in university students' health education.

Time trend and clinical pattern of extrapulmonary tuberculosis in Serbia, 1993-2007.

BACKGROUND/AIM: Increased incidence of extrapulmonary tuberculosis (XPTB) is reported worldwide. Serbia is a country in socio-economic transition period with low-middle HIV prevalence and intermediate-to-low tuberculosis (TB) incidence rate, 100% directly observed treatment (DOT) coverage, and mandatory BCGC vaccination at birth. The aim of the study was to examine the incidence trend and clinical features of XPTB in Serbia during a 15-year period. METHODS: This retrospective observational study included XPTB cases diagnosed in the period between 1st January 1993 and 31st Decembre 2007, according to the reports of the National Referral Institute of Lung Diseases and Tuberculosis in Belgrade and Central Tuberculosis Register. Population estimates with extrapolations were based on 1991 and 2002 census data. RESULTS: While the overall TB incidence rate showed a slight, not significant decreasing trend (p = 0.535), a significant increase was found for XPTB (y = 1.7996 + 0.089x; R2 = 0.4141; p = 0.01). A total of 2,858 XPTB cases (newly diagnosed and 10% relapses) gave an average age specific incidence rate of 2.51/100,000 population (95% confidence interval, SD = 0.6182) with 8.9% annual increase. The male-to-female ratio was 0.54. Lymph nodes were most frequently affected site (48.5%) followed by genitourinary (20.5%), pleural (12%), and osseo-arthicular (10.3%) TB. Treatment outcome was successful in 88.29% of patients (cured and completed), 3.64% died, 5.18% interrupted, 0.57% displaced, and 2.3% unknown. CONCLUSION: Increasing trend of XPTB incidence rate may be a result of increased morbidity due to still present risk factors, possible higher detection rate in Serbia and better notification. A high coverage of newborns with BCG vaccination at birth might contribute to a decreased number and rare XPTB cases in children.

Asthma mortality in Serbia: a 30-year analysis.

INTRODUCTION: The asthma mortality pattern differs among countries. No published evidence is currently available on asthma mortality in a European low-middle-income country in socioeconomic transition. We analyzed the trend of asthma mortality rate in Serbia during the period 1980-2009. MATERIALS AND METHODOLOGY: a descriptive study; source of data: Republic of Serbia Institute of Statistics Death Registry. We analyzed asthma mortality data in the total population, including gender specific analysis, and in the selected ≥ 5-34 year age group. The International Classification of Diseases codes for asthma 493 (1980-1996) and J45 + J46 (1997-2009) were included. Population estimates were based on 1991 and 2002 census data, with extrapolation. RESULTS: The over-all average mortality rate was 7.27 ± 2.14/100,000 inhabitants. The mortality rate peaked in 1981 at 11.3/100,000 but was 4.45/100,000 in the last year of analysis. While both overall mortality rate, as well as gender specific rates, showed clear decreases over the observed 30-year period (average absolute annual decrease of 0.195/100,000 inhabitants, 0.241/100,000 men and 0.149/100,000 women; p < 0.001) with corresponding relative annual decreases of 2.9% for the whole population, 3.0% for men and 2.8% for women, a stable trend of the rate was found in the age group ≥ 5-34 years. Male-to-female ratio was 1.59 ± 0.186, on average and it showed a stable trend over the observed period. CONCLUSION: Although the failure of the health care system during the socioeconomic crisis of the 1990s might have transiently affected asthma mortality rates, the overall mortality rate for the disease shows a favorable decreasing trend.

Health care workers smoke as well--who, how much and why?

In this observational study based on an original questionnaire, we investigated a group of health care workers--smokers employees of a teaching hospital in terms of basic features of tobacco smoking. A total of 137 smokers were asked to answer the questions on voluntary basis and anonymously. Response rate was 70%: a total of 96 participants with valid questionnaires consisted of 93% women, predominantly nurses (71%); mean age 40.5 (range 19-58 years). They started smoking at the age of 20 years on average; 83% started smoking before the age of 23. The mean smoking duration was 19.1 +/- 9.378 years and the average number of the cigarettes smoked per day was 17 +/- 7.90. They smoke 18 +/- 13.55 pack/year; 25% smoke 7.42 pack/year, 25% smoke > 27.5 pack/year. Some 59% had tried and failed to stop smoking while 61% intend to stop smoking (married significantly more intend to quit than singles). The most important reasons for smoking, in a rank order, were: a) habit (35.36%), b) nervousness (12.13%) and c) pleasure (11.11%); boredom and desire were rarely mentioned; only 7 (7.3%) stated addiction. There is an urgent need of continuous medical education on tobacco smoking as an addictive disease, its primary prevention and cure, including professional assistance in smoking cessation.