BusR senses bipartite DNA binding motifs by a unique molecular ruler architecture.

The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.

c-di-AMP hydrolysis by the phosphodiesterase AtaC promotes differentiation of multicellular bacteria.

Antibiotic-producing Streptomyces use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as Streptococcus pneumoniae and Mycobacterium tuberculosis AtaC is monomeric in solution and binds Mn2+ to specifically hydrolyze c-di-AMP to AMP via the intermediate 5'-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-di-AMP promotes interaction between CpeA and the predicted cation/proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in Streptomyces, connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in Streptomyces.

Comparison of prevalence of depression symptoms and history of suicidality in students of medical schools and other study programmes of Charles University.

OBJECTIVES: Medical students represent a group of undergraduate students who are exposed to specific risk factors that may lead to the onset of depression and the occurrence of suicidal ideation. The aim of the article is to present information about the prevalence of symptoms of depression in medical students of different faculties at Charles University, Prague, compared to other students of this university. METHODS: We used a standardized Beck's Inventory scale II (BDI-II) with added specific questions electronically distributed to undergraduate students of the full-time forms of study of all faculties of Charles University. The data collection was anonymous and took place in December 2020. The data were statistically assessed in relation to the occurrence of moderate and severe depression using univariate and multivariable analysis. RESULTS: Moderate and severe depression rate (MSDR) was recorded in 19.6% of medical school students, who returned the questionnaire, compared to 23.4% of students of other faculties (p = 0.001) of Charles University in Prague. Differences in MSDR among students of different faculties of medicine of Charles University were not statistically significant. The total number of previous suicide attempts among respondents was 542, of which 115 were medical students, with the lifetime prevalence of suicide attempts among all students participating in the study 6.96% and in medical students of Charles University 5.73%. CONCLUSIONS: The findings of our study highlight the need for systematic, accessible and timely assistance to university students, both in terms of prevention and early intervention, which can take place at the university level and within the mental healthcare system.

Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides cth GAMP.

The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP-AMP synthase (cGAS) catalyzing the production of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines. Due to the therapeutic potential of this pathway, both the production and the detection of cGAMP via fluorescent moieties for assay development is of great importance. Here, we introduce the paralleled synthetic access to the intrinsically fluorescent, cyclic dinucleotides 2'3'-cth GAMP and 3'3'-cth GAMP based on phosphoramidite and phosphate chemistry, adaptable for large scale synthesis. We examine their binding properties to murine and human STING and confirm biological activity including interferon induction by 2'3'-cth GAMP in THP-1 monocytes. Two-photon imaging revealed successful cellular uptake of 2'3'-cth GAMP in THP-1 cells.

Nationwide observational study of paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) in the Czech Republic.

The worldwide outbreak of the novel 2019 coronavirus disease (COVID-19) has led to recognition of a new immunopathological condition: paediatric inflammatory multisystem syndrome (PIMS-TS). The Czech Republic (CZ) suffered from one of the highest incidences of individuals who tested positive during pandemic waves. The aim of this study was to analyse epidemiological, clinical, and laboratory characteristics of all cases of paediatric inflammatory multisystem syndrome (PIMS-TS) in the Czech Republic (CZ) and their predictors of severe course. We performed a retrospective-prospective nationwide observational study based on patients hospitalised with PIMS-TS in CZ between 1 November 2020 and 31 May 2021. The anonymised data of patients were abstracted from medical record review. Using the inclusion criteria according to World Health Organization definition, 207 patients with PIMS-TS were enrolled in this study. The incidence of PIMS-TS out of all SARS-CoV-2-positive children was 0.9:1,000. The estimated delay between the occurrence of PIMS-TS and the COVID-19 pandemic wave was 3 weeks. The significant initial predictors of myocardial dysfunction included mainly cardiovascular signs (hypotension, oedema, oliguria/anuria, and prolonged capillary refill). During follow-up, most patients (98.8%) had normal cardiac function, with no residual findings. No fatal cases were reported.Conclusions: A 3-week interval in combination with incidence of COVID-19 could help increase pre-test probability of PIMS-TS during pandemic waves in the suspected cases. Although the parameters of the models do not allow one to completely divide patients into high and low risk groups, knowing the most important predictors surely could help clinical management.

A Simple Method for Quantification of Polyhydroxybutyrate and Polylactic Acid Micro-Bioplastics in Soils by Evolved Gas Analysis.

Conventional plastics are being slowly replaced by biodegradable ones to prevent plastic pollution. However, in the natural environment, the biodegradation of plastics is usually slow or incomplete due to unfavorable conditions and leads to faster micro-bioplastic formation. Many analytical methods were developed to determine microplastics, but micro-bioplastics are still overlooked. This work presents a simple method for determining poly-3-hydroxybutyrate and polylactic acid micro-bioplastics in soil based on the thermogravimetry-mass spectrometry analysis of low molecular gases evolved during pyrolysis. For the method development, model soils containing different soil organic carbon contents were spiked with micro-bioplastics. Specific gaseous pyrolysis products of the analytes were identified, while the ratio of their amounts appeared to be constant above the level of detection of the suggested method. The constant ratio was explained as a lower soil influence on the evolution of the gaseous product, and it was suggested as an additional identification parameter. The advantages of the presented method are no sample pretreatment, presumably no need for an internal standard, low temperature needed for the transfer of gaseous products and the possibility of using its principles with other, cheaper detectors. The method can find application in the verification of biodegradation tests and in the monitoring of soils after the application of biodegradable products.

Safe environment management in acute psychiatric wards in the Czech Republic, foundation for recommendations for preventive practice.

OBJECTIVES: Suicides of hospitalized patients present rare but very serious adverse events in healthcare settings. The aim of this article is to describe and analyse the facilities and material equipment of acute psychiatric settings in the Czech Republic and contrast these with recommendations for effective prevention of suicidal behaviour. Since there are currently no universally accepted protocols for risk assessment and prevention of suicides in hospital settings in the Czech Republic, these recommendations draw on international guidelines. Based on the outcomes of our study we provide recommendations for risk management and effective prevention of suicidal behaviour of patients hospitalized in acute care settings. METHODS: In order to describe and analyse the environment of acute psychiatric wards in the Czech Republic we have developed a questionnaire based on international recommendations for risk management and prevention of suicidal behaviour. We also collected data on the prevalence of attempted and completed suicides and their respective methods in these hospitals. RESULTS: We have established that acute psychiatric wards in the Czech Republic operate within insufficient safety regimes, especially with respect to the prevention of suicide by hanging and the accessibility of objects for cutting. Our findings demonstrate that only 75% of the wards are equipped with safety glass, and only 50% of the wards with safety mirrors. Only just over 40% of hospitals have safety door handles and shower heads. CONCLUSION: While it is impossible to entirely eliminate the risk of suicidal behaviour it is possible to manage it. The risk reduction is attainable by providing a safe-proofed environment and minimizing opportunities of suicide attempts by hanging and cutting. In order to effectively prevent suicides, it is essential to increase the awareness of the possibilities of safe proofing of the environment as well as standardization of risk assessment of potential suicidal behaviour of patients.

Newborn screening-detected 21-hydroxylase deficiency: growth pattern is not associated with the genotype.

BACKGROUND: Normalising growth in children with congenital adrenal hyperplasia due to 21- hydroxylase deficiency (21OHD-CAH) requires a long-term maintenance of a fragile balance between hydrocortisone (HC) replacement and androgen suppression. The growth pattern inchildren with 21OHD-CAH diagnosed by clinical symptoms has been evaluated in numerous retrospective studies. The aim of this study was to evaluate growth of patients with 21OHD-CAH detected by newborn screening (NBS), prior to clinical symptoms. METHODS: Nation-wide NBS for 21OHD-CAH was implemented in the Czech Republic in 2006. Since then, 1,317,987 neonates were screened (2006-2017) and 21OHD-CAH was confirmed in 108 patients. Growth was evaluated as height-standard deviation score (SDS) at regular time-points, related to bone age and compared to recent population standards. In 88 patients, available data allowed long-term evaluation of growth, HC and fludrocortisone doses (in half-year intervals), with a median observation period of ten years. RESULTS: Body height in affected children was shorter between years 1-9 of life with a nadir at age 1-3 years. Their height did not differ from general population at the age 10-12 years. There were not found differencies according to 21OHD-CAH severity. CONCLUSIONS: NBS is an effective secondary prevention tool for the early detection of 21OHD-CAH which improves growth patterns. A significant growth deceleration was observed during infancy and early childhood periods but with following height normalization. Growth pattern was not associated with the genotype of 21OHD, if patients have been detected by NBS.

A Click-Chemistry Linked 2'3'-cGAMP Analogue.

2'3'-cGAMP is an uncanonical cyclic dinucleotide where one A and one G base are connected via a 3'-5' and a unique 2'-5' linkage. The molecule is produced by the cyclase cGAS in response to cytosolic DNA binding. cGAMP activates STING and hence one of the most powerful pathways of innate immunity. cGAMP analogues with uncharged linkages that feature better cellular penetrability are currently highly desired. Here, the synthesis of a cGAMP analogue with one amide and one triazole linkage is reported. The molecule is best prepared via a first CuI -catalyzed click reaction, which establishes the triazole, while the cyclization is achieved by macrolactamization.

Epidemiology of rare diseases detected by newborn screening in the Czech Republic.

OBJECTIVES: Presymptomatic detection of patients with rare diseases (RD), defined by a population frequency less than 1 : 2,000, is the task of newborn screening (NBS). In the Czech Republic (CZ), currently eighteen RD are screened: phenylketonuria/hyperphenylalaninemia (PKU/HPA), congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH), cystic fibrosis (CF), medium chain acyl-CoA dehydrogenase deficiency (MCADD), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), very long chain acyl-CoA dehydrogenase deficiency (VLCADD), carnitine palmitoyl transferase I and II deficiency (CPTID, CPTIID), carnitine-acylcarnitine translocase deficiency (CACTD), maple syrup urine disease (MSUD), glutaric aciduria type I (GA I), isovaleryl-CoA dehydrogenase deficiency (IVA), argininemia (ARG), citrullinemia (CIT), biotinidase deficiency (BTD), cystathionine beta-synthase-deficient homocystinuria (CBSD HCU), and methylenetetrahydrofolate reductase deficiency homocystinuria (MTHFRD HCU). The aim was to analyze the prevalence of RD screened by NBS in CZ. METHODS: We examined the NBS programme in CZ from 1 January 2010 to 31 December 2017, which covered 888,891 neonates. Dried blood spots were primarily analyzed using fluorescence immuno-assay, tandem mass spectrometry and fluorimetry. RESULTS: The overall prevalence of RD among the neonate cohort was 1 : 1,043. Individually, 1 : 2,877 for CH, 1 : 5,521 for PKU/HPA, 1 : 6,536 for CF (1 : 5,887 including false negative patients), 1 : 12,520 for CAH, 1 : 22,222 for MCADD, 1 : 80,808 for LCHADD, 1 : 177,778 for GA I, 1 : 177,778 for IVA, 1 : 222,223 for VLCADD, 1 : 296,297 for MSUD, 1 : 8,638 for BTD, and 1 : 181,396 for CBSD HCU. CONCLUSIONS: The observed prevalence of RD, based on NBS, corresponds to that expected, more precisely it was higher for BTD and lower for MSUD, IVA, CBSD HCU, MCADD and VLCADD. Early detection of rare diseases by means of NBS is an effective secondary prevention tool.

Quantitative Analysis of Poly(ethylene terephthalate) Microplastics in Soil via Thermogravimetry-Mass Spectrometry.

The use of plastic materials in daily life, industry, and agriculture can cause soil pollution with plastic fragments down to the micrometer scale, i.e., microplastics. Quantitative assessment of microplastics in soil has been limited so far. Until now, microplastic analyses in soil require laborious sample cleanup and are mostly restricted to qualitative assessments. In this study, we applied thermogravimetry-mass spectrometry (TGA-MS) to develop a method for the direct quantitative analysis of poly(ethylene terephthalate) (PET) without further sample pretreatment. For this, soil samples containing 1.61 ± 0.15 wt % organic matter were spiked with 0.23-4.59 wt % PET bottle recyclate microplastics. dl-Cysteine was used as the internal standard (IS). Sample mixtures were pyrolyzed with a 5 K min-1 ramp (40-1000 °C), while sample mass loss and MS signal intensity of typical PET pyrolysis products were recorded. We found MS signal intensities linearly responding to microplastic concentrations. The most-promising results were obtained with the IS-corrected PET pyrolysis product vinylbenzene/benzoic acid ( m/ z = 105, adj. R2 = 0.987). The limits of detection and quantification were 0.07 and 1.72 wt % PET, respectively. Our results suggest that TGA-MS can be an easy and viable complement to existing methods such as pyrolysis or thermogravimetry-thermal desorption assays followed by gas chromatography/mass spectrometry detection or to spectral microscopy techniques.

Neonatal screening in the Czech Republic: increased prevalence of selected diseases in low birthweight neonates.

Neonates with low birthweight (LBW) represent a vulnerable population. This retrospective study analyzed the birth frequency of diseases detected by neonatal screening (NBS) in normal and LBW neonates in the Czech Republic. Between years 2002 and 2016, the number of screened disorders in the Czech Republic gradually increased from two to 13. Prevalence of screened diseases was calculated for cohorts ranging from 777,100 to 1,277,283 neonates stratified by birthweight. Odds ratio of the association of LBW with each disease was calculated and statistical significance was evaluated using the chi-square test or Fisher's exact test, as appropriate. Three diseases were associated with higher risk of prevalence in LBW neonates, namely congenital hypothyroidism (OR 2.50, CI 1.92; 3.25), cystic fibrosis (OR 2.44, CI 1.51; 3.94), and long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) (OR 7.74, CI 2.18; 27.42).Conclusion: Although the underlying mechanisms are not well understood, results can be hypothesized that LBW (respectively prematurity) may lead to the secondary and often transitory hypothyroidism while cystic fibrosis and LCHADD may manifest already prenatally and result into preterm birth and LBW. What is Known: • The percentage of low birthweight (LBW) neonates in the Czech Republic has been increasing. • Previously published studies reported positive association between LBW and congenital hypothyroidism and cystic fibrosis. What is New: • The association between LCHADD and LBW has not yet been described. • LBW can be the first manifestation of cystic fibrosis and LCHADD.